Keywords: apolipoprotéine a-I (ApoA1), Pichia pastoris, ApoA1-Milano et ApoA1-Paris, chromatographie par mode-mixte, HEA HyperCel, PPA HyperCel, Capto apolipoprotein a-I (ApoA1), Pichia pastoris, ApoA1-Milano and ApoA1-Paris, mixed-mode chromatorgraphy, HEA HyperCel, PPA HyperCel, Capto

de thèse en

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Avant-propos / Acknowledgement

This doctoral thesis has been an exciting journey, over the past three years with more experiences than just experimental data: I won a couple of national fellowships, spent a year in France, learnt to converse fluently in French, got married, published two reasonably good papers, won a couple of presentation awards, wrote grants, and am now on my way to becoming an entrepreneur with my own biotech start-up. I am writing this small note to try and thank every person who has enriched this journey, and have added value and meaning to my thesis and my life.

To begin with, I cannot thank enough Prof. M.A. Vijayalakshmi, Director, CBST, VIT University, who has been my mentor (and mother when the times required), and has shaped me from the little boy that I was to what I am today. None of my milestones would have been possible without her constant support and criticism, which has really shaped me for the world. Viji's energy and enthusiasm even today (while she's 72 years young) is an important daily reminder for me not to slag in my work and other aspects of life.

I wish to convey my sincere thanks to my thesis directors, Prof. Xavier Santarelli (Professor, EA-4135, ENSTBB/IPB, Université de Bordeaux, France) and Prof. Krishnan Venkataraman (Professor, CBST, VIT University, India). Krishnan has been great support with my work, and has provided much advice from his life experiences. The excitement he shows while discussing science and data still inspires me to work more and be more productive! Xavier has been an inspiration on handling multiple things swiftly and his carrying out experiments like a young post-doc always reminds me not to sleep away from the bench. I also thank Dr. Majid Noubhani (Maître de conférences, ENSTBB/IPB, Université de Bordeaux), who has been second to Xavier in helping me with my thesis and in the experimental work, especially with handling P. pastoris. I thank Dr. M. Balasubramanyam (Asst. Director, MDRF, Chennai) and Dr. Suvro Chatterjee (Faculty Scientist, AU-KBC Research Centre, Chennai) who have been kind in acting as my external doctoral committee members and have provided valuable inputs throughout my thesis during my presentations. I also thank Dr. Ayesha Noor, Asst. Professor (Senior), CBST, VIT University, who in addition to being my doctoral committee's internal member also took care of coordinating with the research office and in organising the logistics for presentations, which made life at CBST very easy. I thank Dr. Rajasekar Prasanna (now with ThermoScientific, India), Dr. Stephane Chaignepain (CBMN, UMR5248, Université de Bordeaux) and Ms. Goldy Sachdev (CBST, VIT University) for their support in my experimental work for proteomic analyses and expression in protease deficient strain of P. pastoris.

I must extend my gratitude to my friends and colleagues at both research teams (EA-4135, ENSTBB/IPB, Université de Bordeaux, France and CBST, VIT University, Vellore, India) for their support and cooperation throughout this journey. (Miller, 1978).

Il est généralement accepté que les lipoprotéines de haute densité (High Density Lipoprotein, HDL) permet de réduire le risque de maladies cardio-vasculaires, car il se déplace le cholestérol au foie par le transport inverse du cholestérol (Reverse Cholesterol Transport, RCT) (Tall, Costet, & Wang, 2002). En plus, le HDL contribue également au bien-être général du système cardio-vasculaire grâce à ses propriétés anti-inflammatoires, anti-oxydantes et anti-thrombotiques [START_REF] Assmann | HDL Cholesterol and Protective Factors in Atherosclerosis[END_REF]. On pense que ces effets protecteurs des HDL être médiée principalement par l'apolipoprotéine AI (ApoA1) qui est le composant protéique majeur des HDL [START_REF] Heinecke | The Protein Cargo of HDL: Implications for Vascular Wall Biology and Therapeutics[END_REF].

R.1.2. TRANSPORT INVERSE DU CHOLESTEROL

L'ApoA1 favorise l'efflux de cholestérol des tissus vers le foie (Oram, 2003) (Fig.

R.1). ApoA1 prend cholestérol des cellules à travers l'ABCA1 (ATP-Binding Cassette A1) et forme des particules de HDL naissantes. Les particules HDL plus matures lors de l'activation de la Lécithine-cholestérol acyltransférase (LCAT) qui convertir le cholestérol à des esters de cholestérol [START_REF] Vanloo | LCAT Activation Properties of Apo A-I CNBr Fragments and Conversion of Discoidal Complexes into Spherical Particles[END_REF]. Ces esters de cholestérol de HDL sont échangés avec des triglycérides provenant d'autres lipoprotéines (LDL/VLDL) par l'action de la protéine de transfert des esters de cholestérol (Cholesteryl Ester Transfer Protein, CETP). Le transport inverse du cholestérol est complétée par le dépôt d'esters de cholestérol dans le foie, soit directement par Scavenging récepteur B1 (SR-B1) ou par LDL et le LDLrécepteur [START_REF] Lewis | New Insights Into the Regulation of HDL Metabolism and Reverse Cholesterol Transport[END_REF]. On sait que des niveaux élevés dans le plasma de l'ApoA1 contribue à la réduction des risques cardio-vasculaires chez les humains [START_REF] Gordon | High-Density Lipoprotein Cholesterol and Cardiovascular Disease. Four Prospective American Studies[END_REF], ce qui rend l'ApoA1 du potentiel thérapeutique. En outre, deux variants naturels de ApoA1 : ApoA1-Milano et ApoA1-Paris ont été signalés dans les populations qui ont réduit risque d'athérosclérose (Alexander et al., 2009). Les variantes Milano et de Paris sont caractérisées par des mutations ponctuelles au R173C et R151C respectivement. L'ApoA1 de type sauvage ne contient pas de résidus de cystéine, et donc l'introduction de ces mutants cystéine dans leur permet de former des homo-dimères à liaison disulfure sur le HDL [START_REF] Klon | Molecular Belt Models for the Apolipoprotein A-I Paris and Milano Mutations[END_REF].

R.1.3. LES OBJECTIVES

Dans ce travail, nous avons mis en place un processus simplifié pour générer l'ApoA1 recombinante chez Pichia pastoris et de la purifier par chromatographie en mode mixte.

Le système d'expression choisi pour cette étude est la levure méthylotrophe Pichia pastoris, en raison d'un certain nombre d'avantages qu'elle pose : la capacité d'atteindre des niveaux élevés d'expression de protéines hétérologues (Cereghino & Cregg, 2000;[START_REF] Sreekrishna | Strategies for Optimal Synthesis and Secretion of Heterologous Proteins in the Methylotrophic Yeast Pichia Pastoris[END_REF], la capacité à sécréter des protéines hétérologues dans le milieu [START_REF] Brake | Alpha-Factor-Directed Synthesis and Secretion of Mature Foreign Proteins in Saccharomyces Cerevisiae[END_REF], et étant «généralement considéré comme sûr" (Generally Regarded As Safe, GRAS) micro-organismes [START_REF] Klein | High-Level Secretion of a Wheat Lipid Transfer Protein inPichia Pastoris[END_REF]. La surexpression de protéines hétérologues chez P. pastoris est atteinte par le promoteur AOX1 qui est activé dans des conditions de carbone faim [START_REF] Daly | Expression of Heterologous Proteins in Pichia Pastoris: A Useful Experimental Tool in Protein Engineering and Production[END_REF].

Après l'expression, l'ApoA1 recombinante a été ensuite purifié par Chromatographie en mode mixte. Chromatographie par mode mixte ou « multimodal » sont généralement émis l'hypothèse d'agir par une combinaison d'interactions électrostatiques et hydrophobes (Chung, Freed, Holstein, McCallum, & Cramer, 2010).

Donc, les objectifs suivants sont envisagés dans ce travail de thèse:

• Clonage et expresion de type sauvage l'ApoA1 recombinante (rhApoA1) dans P.

pastoris X-33

• Purification de rhApoA1 de la culture de P. pastoris, et scale-up de la purification à grande échelle avec l'adsorption a lit expansée (Expanded Bed Adsorption, EBA)

• Le clonage, l'expression et la purification de rhApoA1 chez P. pastoris SMD-1168

(souche de P. pastoris déficiente en protéase)

• Génération de variantes de l'ApoA1: Milano Après vérification de l'expression, la milieu de P. pastoris contenant rhApoA1 a été utilisé pour les expériences de purification. Un certain nombre de méthodes de purification publiés ont été testés pour leur aptitude à récupérer rhApoA1 exprimée dans P. pastoris [START_REF] Feng | High Yield and Secretion of Recombinant Human Apolipoprotein AI in Pichia Pastoris[END_REF][START_REF] Aurélio | Cloud Point Method Applied to the Apolipoprotein A-I Extraction from Human Plasma and Its Identification by Tandem Mass Spectrometry[END_REF]. Ces méthodes ont été conservés en tant que méthodes de référence pour évaluer l'efficacité des procédés de purification développé dans cette étude.

Deux méthodes de chromatographie en mode mixte ont été testés pour leur capacité à capturer rhApoA1 directement à partir du milieu d'expression de P. La profil de purification de la rhApoA1 chez P. pastoris SMD1168 à l'aide Capto™ MMC a été similaires de purification profil obtenu en purifiant rhApoA1 de P.

pastoris X-33.

En analyse par spectrométrie en mass, les rhApoA1 purifiée à partir de deux souches des P. pastoris, il a été observé qu'il n'y avait pas de différence significative entre les poids moléculaires des deux souches de rhApoA1 (Fig. [START_REF] Reddy | Emerging Epidemic of Cardiovascular Disease in Developing Countries[END_REF]. Owing to the rising number of CVD cases, India is slated to become the world's CVD capital within the next ten years [START_REF] Gupta | Epidemiology and Causation of Coronary Heart Disease and Stroke in India[END_REF].

Atherosclerosis is one of the major components of CVD, and is characterised by the hardening of arteries due to invasion and accumulation of macrophages and subsequent build-up of cholesterol, lipids and lipoproteins [START_REF] Epstein | Atherosclerosis -An Inflammatory Disease[END_REF]. Advanced stages of atherosclerosis often occlude blood flow causing several mortal conditions such as myocardial infarction, cardiovascular stroke, or form emboli that could affect other tissues. One of the major causative reasons for the progression of atheroma is the dysregulation of cholesterol metabolism in the body [START_REF] Assmann | HDL Cholesterol and Protective Factors in Atherosclerosis[END_REF].

CHOLESTEROL AND ITS METABOLISM

Cholesterol is a sterol molecule, which is an essential structural component of higher eukaryotic cells, in addition to being a precursor molecule for the biosynthesis of steroid hormones, bile acids, etc. [START_REF] Hanukoglu | Steroidogenic Enzymes: Structure, Function, and Role in Regulation of Steroid Hormone Biosynthesis[END_REF]. Cholesterol The cholesterol molecule contains a hydroxyl group that enables it to interact with the polar heads of membrane phospholipids and sphingolipids; whereas the bulky steroid and hydrocarbon chain sink into the membrane along with they hydrophobic fatty acid chains of lipids [START_REF] Yeagle | Modulation of Membrane Function by Cholesterol[END_REF]. This ability of cholesterol to blend in with membrane lipids enables it to provide fluidity to the cell membrane when required. In addition to providing membrane fluidity, cholesterol is also a precursor molecule for the synthesis of steroid hormones and bile acid [START_REF] Berg | Crystal Structure of Truncated Human Apolipoprotein A-I Suggests a Lipid-Bound Conformation[END_REF].

The average cholesterol intake is approximately 400mg per day, with primary sources being de novo biosynthesis and diet. About 50% of the cholesterol from dietary sources is actually absorbed (the balance is directly excreted), and hence there is a bulk production through biosynthesis pathways for catering to the body's cholesterol requirements [START_REF] Lehninger | Apolipoprotein A-I Is a Selective Target for Myeloperoxidase-Catalyzed Oxidation and Functional Impairment in Subjects with Cardiovascular Disease[END_REF].

As cholesterol is only mildly soluble in water, it is primarily transported in blood through protein carriers called lipoproteins, which are complex structures with a hydrophilic exterior (with polar lipid heads and proteins) and an apolar core (explained in greater detail in Section 1.2). Lipoproteins are classified based on their density, and vary in protein compositions and functions.

LIPOPROTEINS

Lipoproteins are complex biological assemblies of lipids and proteins that enable the transport of fatty acids and cholesterol through water inside and outside cells [START_REF] Jonas | Lipoprotein Structure[END_REF]. The proteins enable the emulsification of lipid particles, facilitating the smooth movement of cholesterol and triglycerides through blood.

STRUCTURE AND CLASSIFICATION OF LIPOPROTEINS

Lipoprotein molecules vary from discoidal to spherical forms, with a hydrophobic core and a hydrophilic surface [START_REF] Jonas | Lipoprotein Structure[END_REF].

As marked in Fig. 1.2, the proteins and the hydrophilic head of lipid molecules form the polar outer shell encapsulating the hydrophobic core made of cholesterol, fatty acids and triglycerides. The most important protein component of lipoproteins is apoproteins. Apoproteins bind to lipids to form nascent lipoproteins [START_REF] Jackson | Lipoprotein Structure and Metabolism[END_REF]. There are a number of apoproteins associated with various sizes of lipoproteins; for example, apolipoprotein B associates with LDL and chylomicrons and apolipoprotein A associates with HDL. Lipoproteins can be classified based on three different parameters:

HIGH-DENSITY LIPOPROTEINS (HDL)

The High Density Lipoprotein, or HDL, is the densest and smallest of lipoproteins. HDL particles range from 7 to 12nm in diameter, and from 1.063 to 1.25 g/ml in density (P. Barter et al. 2003). Compared to other lipoproteins, HDLs have the highest proportion of proteins (>50%) relative to their lipid content. The hydrophobic core of HDL is mainly composed of cholesterol/cholesterol-esters and a small amount of triglyceride [START_REF] Rifai | Handbook of Lipoprotein Testing[END_REF]. The major lipid composition of HDL includes phospholipids (50%), cholesteryl esters (30%), free cholesterol (10%) and triglycerides (10%).

One of the key characteristics of HDL is their heterogeneity: in size, density and apolipoprotein composition. HDLs are classified into five sub-populations according to their size (row 3, Table 1.1), and two major subfractions based on their density (row 4, Table 1.1). Furthermore, they are also classified based on their apolipoprotein composition into those that contain only ApoA1 (A-I HDL)

and ones that contain both ApoA1 & ApoA2 (A-I/A-II HDL), highlighted in row 2 (Table 1.1).

HDL is generally perceived as cardio-protective, primarily due to its role in Reverse Cholesterol Transport (RCT), in addition to anti-oxidant, antiinflammatory, anti-apoptotic, anti-thrombotic and anti-platelet activating effects [START_REF] Tabet | High-Density Lipoproteins, Inflammation and Oxidative Stress[END_REF].

REVERSE CHOLESTEROL TRANSPORT (RCT)

The process by which cholesterol is moved from peripheral tissues to the liver for catabolism is known as Reverse Cholesterol Transport (RCT, Fig. 1.4). RCT is the major cardioprotective function of the HDL particle [START_REF] Fielding | Molecular Physiology of Reverse Cholesterol Transport[END_REF]. Briefly, either lipid-free ApoA1 or lipid-poor pre-β HDL particles (from liver or intestine) accept cholesterol from macrophages through the effluxor ABCA1

(ATP-Binding Cassette A1) to form discoidal HDL particles. This discoidal HDL particle accepts further cholesterol and trigliceride molecules to form a spherical HDL molecule. The key step in RCT is the activation of HDL by Lecithin-Cholesterol Acyl Transferase (LCAT), an enzyme that catalyses the conversion of cholesterol to cholesterylester, resulting in the maturation of HDL [START_REF] Vanloo | LCAT Activation Properties of Apo A-I CNBr Fragments and Conversion of Discoidal Complexes into Spherical Particles[END_REF]. From the mature HDL, there are two routes for the cholesterol to reach the liver: one directly through Scavenging Receptor B1 (SR-B1); second through the LDL to LDL-receptor. The exchange of cholesterol/cholsesterylester/triglycerides is achieved by the action of Cholesteryl Ester Transfer Protein (CETP), which is a key actor in maintaining the ratio of HDL to LDL in the blood (P. J. Barter et al. 2003). Once cholesterol/ cholesterylester reaches the liver, it is subsequently converted to bile acid and excreted via feces.

The RCT represents one of the key pathways by which cholesterol homeostasis is maintained in the body, and a number of interventions have been proposed to intervene/enhance various stages of this pathway, which will be discussed in greater detail in the following sub-section.

LIPOPROTEIN RATIO & CARDIOVASCULAR WELLBEING

Over the decades, several epidemiological studies have demonstrated that increasing levels of HDL are inversely correlated to risk of CHD/CVD and atherosclerosis [START_REF] Assmann | HDL Cholesterol and Protective Factors in Atherosclerosis[END_REF]P. Barter et al. 2003;[START_REF] Kannel | Serum Cholesterol, Lipoproteins, and the Risk of Coronary Heart DiseaseThe Framingham Study[END_REF]. The Framingham Study was the first to report a correlation between serum cholesterol levels and coronary heart disease risk (William B. [START_REF] Kannel | Serum Cholesterol, Lipoproteins, and the Risk of Coronary Heart DiseaseThe Framingham Study[END_REF], classifying cardiovascular risk based on the ratio of total cholesterol to HDL-cholesterol (W. B. [START_REF] Kannel | High-Density Lipoproteins: Epidemiologic Profile and Risks of Coronary Artery Disease[END_REF]. This has been subsequently followed by numerous epidemiological and clinical studies across the globe, resulting in the inclusion of HDL-C in the list of risk-predictors for atherosclerosis and cardiovascular disorders [START_REF] Assmann | HDL Cholesterol and Protective Factors in Atherosclerosis[END_REF]. It is estimated that a 1mg/dl increase in HDL-cholesterol correlates to a 2-3% reduction in the risk of cardiovascular events [START_REF] Gordon | High-Density Lipoprotein Cholesterol and Cardiovascular Disease. Four Prospective American Studies[END_REF]).

On the other hand, high levels of LDL and LDL-C (LDL cholesterol) were correlated with an increased risk of CHD/CVD. However, risk factors based on absolute values of LDL/LDL-C weren't sufficient to accurately predict disposition to CHD, especially for patients with intermediate risk [START_REF] Superko | Lipid Management to Reduce Cardiovascular Risk: A New Strategy Is Required[END_REF].

Subsequently, ratios of total cholesterol (TC) to HDL cholesterol (HDL-C), and LDL-C to HDL-C were considered to provide a more reliable risk assessment.

Typical risk levels and target levels to be achieved to bring down risk have been summarised in Table 1.2.

Table 1.2. Risk categories and target levels for CHD [START_REF] Millán | Lipoprotein Ratios: Physiological Significance and Clinical Usefulness in Cardiovascular Prevention[END_REF]. ApoB/ApoA-I >1.0 >0.9 <0.9 <0.8 >0.8 >0.7 <0.7 <0.6

Ratio

A key point to note in table 1.2 is the distinction between primary and secondary prevention ratios. In persons without established CHD, risk levels and target ratios are as defined under primary prevention. Once a CHD event has taken place, there is a significant increase in cardiovascular risk, and hence the target levels for various parameters are much lower than for primary prevention persons [START_REF] Grundy | Assessment of Cardiovascular Risk by Use of Multiple-Risk-Factor Assessment Equations A Statement for Healthcare Professionals From the American Heart Association and the American College of Cardiology[END_REF].

Although absolute values and ratios of various lipoproteins and cholesterol prove to be a reasonable indicator for cardiovascular risk, there have been several exceptions to it. Some studies have reported the occurrence of CVD in certain individuals despite high levels of HDL [START_REF] Manninen | Joint Effects of Serum Triglyceride and LDL Cholesterol and HDL Cholesterol Concentrations on Coronary Heart Disease Risk in the Helsinki Heart Study. Implications for Treatment[END_REF].

Furthermore, reports at the beginning of this millennium showcased the propensity of HDL and its major protein component apolipoprotein A-I to undergo oxidation [START_REF] Lehninger | Apolipoprotein A-I Is a Selective Target for Myeloperoxidase-Catalyzed Oxidation and Functional Impairment in Subjects with Cardiovascular Disease[END_REF][START_REF] Panzenböck | Oxidation of Methionine Residues to Methionine Sulfoxides Does Not Decrease Potential Antiatherogenic Properties of Apolipoprotein A-I[END_REF][START_REF] Shao | Methionine Oxidation Impairs Reverse Cholesterol Transport by Apolipoprotein A-I[END_REF].

Oxidised ApoA1/HDL showed reduced ability in cholesterol binding and LCAT activation. In addition, for several decades, studies have shown the presence of better quality ApoA1/HDL in certain population, through naturally occurring mutations (Alexander et al. 2009;[START_REF] Weisgraber | Apolipoprotein A-IMilano. Detection of Normal A-I in Affected Subjects and Evidence for a Cysteine for Arginine Substitution in the Variant A-I[END_REF]. This brought in a paradigm shift in thinking that the mere quantity of HDL wasn't sufficient for determining cardiovascular risk, but the quality of the HDL particle also had to be considered for determining risk. Subsequently, a number of diagnostic targets have been envisaged, which have been detailed in the next section (Smith 2010).

APOLIPOPROTEIN A-I

Human Apolipoprotein A-I (ApoA1) is the major protein component of the HDL, constituting about 70% of the total HDL protein [START_REF] Rogers | Truncation of the Amino Terminus of Human Apolipoprotein A-I Substantially Alters Only the Lipid-Free Conformation †[END_REF]). In addition to providing major structural support to HDL particles, it also plays two important functional roles: the extraction of cholesterol from peripheral tissues by interacting with ABCA1; and in the activation of LCAT, which is a key factor in reverse cholesterol transport [START_REF] Berg | Crystal Structure of Truncated Human Apolipoprotein A-I Suggests a Lipid-Bound Conformation[END_REF]. This section will cover all aspects of ApoA1, its life cycle, structure, variants and sensitivity to modifications.

PROTEINS OF HDL

The High Density Lipoprotein has been described to be cardio protective in nature, primarily owing to the positive effects exhibited by its various proteins [START_REF] Heinecke | The Protein Cargo of HDL: Implications for Vascular Wall Biology and Therapeutics[END_REF]). In addition to the major protein ApoA1, other HDL proteins such as ApoA2, ApoD, ApoE, paraoxonase 1 (PON1), are also involved in various functions of HDL. Some of the key functions of the HDL enzymes are detailed in Table 1.3. As is evident from Table 1.3, ApoA1 is a key effector of major protective functions of HDL, especially in reverse cholesterol transport [START_REF] Fielding | Molecular Physiology of Reverse Cholesterol Transport[END_REF].

LIFE CYCLE OF ApoA1

The overall life cycle of ApoA1 is closely linked to that of the HDL molecule itself.

ApoA1 molecules are produced by the liver or intestines, or are released from lipolysed VLDL and chylomicrons (von [START_REF] Eckardstein | High Density Lipoproteins and Arteriosclerosis : Role of Cholesterol Efflux and Reverse Cholesterol Transport[END_REF].

These free ApoA1 molecules accept cholesterol from peripheral cells through ABCA1 to form discoidal HDL molecules. They move through the RCT as explained in section 1.2.3, and are eventually cleared in the liver at the end of the reverse cholesterol transport, wherein they are recycled [START_REF] Fielding | Molecular Physiology of Reverse Cholesterol Transport[END_REF].

The ApoA1 protein is expressed as a single polypeptide chain of 243 amino acids, with a 24 amino acid signal peptide sequence for its secretion. Once in circulation, it self-associates into a 4-helix bundle linked via their hydrophobic faces to transport cholesterol, triglycerides and lipids [START_REF] Lewis | New Insights Into the Regulation of HDL Metabolism and Reverse Cholesterol Transport[END_REF].

STRUCTURE OF ApoA1

ApoA1 is a 28kDa monomer that is synthesised by the liver and intestine. The This structure was later confirmed by a subsequent study, revealing addition details. The structure of ApoA1 further showed a solvent-exposed loop from residues 159 to 180, which plays a key role in activation of LCAT [START_REF] Wu | The Refined Structure of Nascent HDL Reveals a Key Functional Domain for Particle Maturation and Dysfunction[END_REF]).

ApoA1 VARIANTS

The ApoA1 gene has been well documented to have several polymorphisms: more than forty naturally occurring variants of ApoA1 have been reported in literature [START_REF] Matsunaga | Apolipoprotein A-I Mutations[END_REF]. Some of the variants have been listed in Table 1.4. et al. 2000). Both these variants have been reported in populations where people had a healthy cardiovascular system despite low HDL levels [START_REF] Rocco | Structural and Dynamic Features of Apolipoprotein A-I Cysteine Mutants, Milano and Paris, in Synthetic HDL[END_REF]).

An important point to note is that wild type ApoA1 contains no cysteine residues, and hence these cysteine variants are hypothesised to be more stable due to formation of homodimers through disulphide bridges [START_REF] Klon | Molecular Belt Models for the Apolipoprotein A-I Paris and Milano Mutations[END_REF]. Hence, in this thesis work, special focus has been laid on the generation of these two ApoA1 cysteine variants, to enable their future applications in therapeutic areas.

MODIFICATIONS ON ApoA1

In addition to naturally occurring variations, ApoA1 is also highly susceptible to modifications under oxidising conditions. Myeloperoxidase (MPO), a defensive enzyme, is expressed by neutrophil granulocytes to fight bacterial infections [START_REF] Klebanoff | Myeloperoxidase: Friend and Foe[END_REF]. However, elevated levels of MPO have been reported in atherosclerotic plaques, leading to a number of modifications on HDL/ApoA1, thereby rendering them dysfunctional [START_REF] Smith | Dysfunctional HDL as a Diagnostic and Therapeutic Target[END_REF]. Table 1.5 lists some of the characterised modifications on ApoA1 by MPO, along with functional consequences that have been reported so far. Among the residues affected by MPO-oxidation, Tryptophan-modifications have been shown to be most consequential. Subsequently, an MPO-mediated oxidation-resistant mutant of ApoA1 was generated by substituting the four Trp residues with Phe residues [START_REF] Peng | Apolipoprotein A-I Tryptophan Substitution Leads to Resistance to Myeloperoxidase-Mediated Loss of Function[END_REF].

Despite extensive studies on the effects of oxidation on wild type ApoA1, there is High levels of ApoA1 have been correlated with a decreased risk of atherosclerosis, and it has long since been hypothesised that elevating plasma ApoA1 levels could lead to regression of atherosclerosis [START_REF] Fazio | Apolipoprotein AI as Therapy for Atherosclerosis: Does the Future of Preventive Cardiology Include Weekly Injections of the HDL Protein?[END_REF].

In addition to the above main function, ApoA1 has recently also been suggested

to be used during in other therapeutic interventions like cancer therapy [START_REF] Su | Apolipoprotein A-I (apoA-I) and apoA-I Mimetic Peptides Inhibit Tumor Development in a Mouse Model of Ovarian Cancer[END_REF]. Keeping in mind the vast diagnostic and therapeutic applications of ApoA1, this thesis focuses on the development of an integrated process for the generation of wild type and variants of ApoA1 in a suitable recombinant proteinproducing host. insulin was the first heterologous protein produced in the laboratory in 1977 [START_REF] Porro | Production of Recombinant Proteins and Metabolites in Yeasts[END_REF]. Several expressions systems are currently being used for the expression of heterologous proteins: like bacterial prokaryotic expression systems (by far E. coli) and eukaryotic systems (yeast and mammalian expressions systems). In addition, plant systems and transgenic animals are being studied for the expression of recombinant therapeutics despite their difficulties, extreme high-cost and ethics involved in their production.

BACTERIAL EXPRESSION SYSTEMS

The bacterial expression system Escherichia coli is the most exploited system for the expression of recombinant proteins. E. coli is favoured as a host for the expression of heterologous proteins for a number of reasons: short doubling time, well established genome, ease of manipulation, GRAS (Generally Regarded

As Safe) status, the ability to scale-up production to industrial scale at minimal costs [START_REF] Makrides | Strategies for Achieving High-Level Expression of Genes in Escherichia Coli[END_REF]. However, plasmid instability and the lack of complex post translational modification machinery like glycosylation limit their use [START_REF] Porro | Production of Recombinant Proteins and Metabolites in Yeasts[END_REF]).

EUKARYOTIC EXPRESSION SYSTEMS

The yeasts Saccharomyces cerevisiae and Pichia pastoris have been developed into highly successful systems for the expression of heterologous proteins [START_REF] Cereghino | Heterologous Protein Expression in the Methylotrophic Yeast Pichia Pastoris[END_REF]. The issue of plasmid instability often faced with prokaryotic systems is overcome in eukaryotes due to stable integration of the gene of interest into the host genome, which is usually achieved through homologous recombination. The ability to grow yeasts in simple, defined media to high cell densities and their ability to perform post-translational modifications make them favourites for the expression of recombinant proteins [START_REF] Demain | Production of Recombinant Proteins by Microbes and Higher Organisms[END_REF]. Nevertheless, hyper-mannosylation remains as a major drawback while working with proteins that require glycosylation. As the target protein in this work (ApoA1) has no glycosylation sites, this would not be affected by expressing in a lower eukaryotic host like yeast.

Pichia pastoris EXPRESSION SYSTEM

The Pichia pastoris expression system serves as a suitable system for the costeffective production of value added proteins. Numerous heterologous proteins have been successfully expressed by the Pichia Pastoris expression system [START_REF] Cereghino | Heterologous Protein Expression in the Methylotrophic Yeast Pichia Pastoris[END_REF]. The P. pastoris system is also generally regarded as being faster, easier, and less expensive to use than expression systems derived from higher eukaryotes such as insect and mammalian tissue culture cell systems and usually gives higher expression levels [START_REF] Klein | High-Level Secretion of a Wheat Lipid Transfer Protein inPichia Pastoris[END_REF][START_REF] Sreekrishna | Strategies for Optimal Synthesis and Secretion of Heterologous Proteins in the Methylotrophic Yeast Pichia Pastoris[END_REF]. Overexpression of heterologous proteins in P. pastoris is achieved by the AOX1 promoter, which is activated under carbon starvation [START_REF] Daly | Expression of Heterologous Proteins in Pichia Pastoris: A Useful Experimental Tool in Protein Engineering and Production[END_REF]. The alcohol oxidase enzyme is encoded for by two genes: AOX1 and AOX2. Most AOX in methanol-grown Pichia pastoris cells is coded for by the AOX1 gene (J. [START_REF] Cregg | Pichia Pastoris as a Host System for Transformations[END_REF]. Hence, transformation of gene of interest into the Pichia genome at the AOX1 gene provides the potential to overexpress heterologous proteins (J. [START_REF] Cregg | Pichia Pastoris as a Host System for Transformations[END_REF].

As the P. pastoris expression vector does not contain a yeast origin of replication, it is essential for the integration of the expression cassette into the host genome.

This recombination is possible through the AOX1 or aox1 loci at the 5'AOX1 Once the expression cassette of the target gene has integrated into the host genome, expression is achieved through carbon starvation and subsequent activation of the AOX1 promotor resulting in the expression of heterologous proteins.

GOAL AND BROAD OBJECTIVES

This thesis has focused on the development of an integrated process for the efficient generation of wild type and variants of human apolipoprotein A-I in Pichia pastoris.

The following objectives outline the research methodology that has been envisaged in this thesis work:

1. Cloning, expression of wild type rhApoA1 in P. pastoris X-33

INTRODUCTION TO CLONING IN P. pastoris

The methylotrophic yeast Pichia pastoris has long since been explored for the heterologous expression of proteins through the alcohol oxidase promoter [START_REF] Cereghino | Heterologous Protein Expression in the Methylotrophic Yeast Pichia Pastoris[END_REF]. A number of Pichia pastoris expressed recombinant products like insulin and hepatitis-B vaccine have already hit the Indian market [START_REF] Shekhar | Pichia Power: India's Biotech Industry Puts Unconventional Yeast to Work[END_REF]. It is hence a very suitable host for the overexpression and production of recombinant ApoA1, as envisaged in this thesis.

Several expression vectors for P. pastoris are commercially available with

Invitrogen, with most of them containing a unique multiple cloning site, resistance to the antibiotic Zeocin and signal peptides for secretory expression.

The vector pPICZα is one of the widely used vectors containing all the above described features, and has been used in the present study too (James M. Cregg et al. 2009, 13). The functioning of AOX1 promoter has already been elucidated in section 1.4.5.

EXPERIMENTAL 2.2.1. MATERIALS

The construct containing human ApoA1 gene was purchased from Open Biosystems (EHS1001-5646018). Oligonucleotides for PCR amplification and cloning were custom-ordered from Sigma-Aldrich (Bangalore, India). All chemicals and reagents for expression and purification were from Sigma-Aldrich (Bangalore, India or Lyon, France) and BD Biosciences (Le Pont de Claix, France).

Expression studies in benchtop bioreactors were carried out on Sartorius-Stedim Biostat B-plus bioreactors monitored by MCSF Win data acquisition software (Sartorius), DNA sequencing was performed through Millegen (Labège, France).

The P. pastoris strain X-33 and vector pPICZαA were kind gifts from Prof. Rajan R. Dighe, Department of Molecular Reproduction, Development and Genetics (MRDG), Indian Institute of Science, Bangalore, India.

CLONING OF rhApoA1 GENE INTO pPICZαA

The sequence corresponding to human ApoA1 was amplified by Polymerase Chain Reaction using the primers 5'ATGAATTCGATGAACCCCCCCAGAGC3' and 5'ATGCGGCCGCTCACTGGGTGTTGAG3' to introduce EcoRI and NotI sites respectively to facilitate cloning. PCR was carried out using 35 cycles of denaturation (94°C for 30"), annealing (56°C for 30"), extension (72°C for 1'), followed by a final extension at 72°C for 10 mins. The reverse primer introduced a stop codon at the end of the gene to prevent expression of the optional hexahistidine tag in the vector. The amplified fragment was cloned into the Pichia pastoris expression vector pPICZαA downstream of the AOX1 promoter and the resulting recombinant DNA construct (pPICZα-ApoA1) was verified by DNA sequencing using AOX1-specific primers by Millegen (Labège, France).

TRANSFORMATION INTO P. pastoris

The plasmid pPICZα-ApoA1 was linearized using PmeI and electroporated into competent Pichia pastoris X-33 cells, as per the manufacturer's instructions. To the transformed cells, 1ml ice-cold 1M sorbitol was added, and the cells were allowed to stand at 30°C for 30 mins, after which they were plated on YPDS plates containing 200µg/ml Zeocin™ for selection. The plates were incubated at 30°C for selection of transformants, and clones exhibiting high resistance to Zeocin™ (up to 2 mg/ml) were taken further for genomic DNA and subsequent expression analysis.

COLONY PCR ANALYSIS

In order to confirm the integration of the recombinant DNA construct pPICZα-ApoA1 into the Zeocin-resistant P. pastoris clones, Colony-PCR was performed on select transformed clones. The primers and PCR conditions were identical to that used to amplify the ApoA1 gene for cloning (Section 2.2.2). Following PCR, the various reactions were analysed by 0.8% agarose gel electrophoresis and stained with SYBR Safe stain.

EXPRESSION STUDIES ON FLASK CULTURES

Several clones of Pichia pastoris transformed with pPICZα-ApoA1 that exhibited high resistance to Zeocin were screened in shake flask for screening for highlevels of expression of rhApoA1.

From the YPD plate containing 2mg/mL Zeocin, five clones were selected for preliminary expression studies, along with a negative control (Pichia pastoris transformed with just the vector pPICZαA without insert). Single colonies of each clone were inoculated independently in 10ml BMGY containing 200 μg/ml Zeocin as a preculture. After overnight growth at 30°C and 200 rpm, the precultures were inoculated into 140 ml of fresh BMGY (buffered glycerol complex media). After about 24 hrs, when the cells had grown to a reasonable quantity (based on their OD at 600nm), they were centrifuged and resuspended in 150 ml of BMMY (buffered methanol complex media). Induction was carried out for a total of 5 days; with methanol being replenished every 24 hrs at 0.5%.

At the end of the induction, comparative day-wise expression of select clones along with the control were analysed by SDS-PAGE and Western blotting.

EXPRESSION IN 2L BENCHTOP BIOREACTOR

Subsequent to preliminary expression studies on shake flasks, the best expressing clone was taken further for scale-up in a 2 l benchtop bioreactor.

First, a single colony was inoculated and grown in baffled flasks containing 100 ml of buffered glycerol-complex medium (BMGY, 100mM potassium phosphate buffer, pH 6.0, 13.4 g/L YNB, 4x10 -4 g/L biotin, 10 g/L glycerol and 150 µg/mL Zeocin™) up to an OD (600nm) of 4-8, and this was inoculated in 2L of BMGY in a BIOSTAT® Bplus laboratory 2l bioreactor with a starting OD600 of about 0.3. The temperature and pH were maintained at 30°C and 6.0 respectively, and dissolved oxygen level was maintained at 15% saturation by regulating aeration and agitation in a cascade manner. After complete consumption of glycerol in the medium (verified by standard glycerol assay; data not shown), a methanol fedbatch phase was initiated by adding methanol every 12 hrs to a final concentration of 0.5%. Samples were drawn every 24 hrs to follow the growth profile and protein production. After 120 hrs of induction, the culture was harvested and the cell-free broth was frozen at -80°C.

SDS-PAGE AND WESTERN BLOTTING

Protein samples were analysed by 12% SDS-PAGE, typically under reducing conditions with DTT. In order to probe for rhApoA1 (western blotting), the proteins from the acrylamide gel were transferred to a 0.45 µm nitrocellulose membrane using Transblot SD transfer apparatus (Bio-Rad, France) according to the manufacturer's instructions. The membrane was blocked with PBST containing 3% skimmed milk, and probed with goat anti-humanApoA1 antiserum (part of an ApoA1 immunoturbidometry-based detection kit, Apolipoprotein A1 FS, Cat. No. 17102, DiaSys, France) followed by rabbit antigoat IgG conjugated to HRP (Jackson ImmunoResearch, PA, USA), and visualised by Opti-4CN colorimetric detection kit (Bio-Rad, France). In order to reduce the background generated by cross-reactivity of the antibodies in the primary antiserum (goat anti-humanApoA1) to native P. pastoris proteins, 100 µg of lyophilized P. pastoris control broth (without transformation of any vector) was added to the primary antiserum solution during blotting.

RESULTS

CLONING OF rhApoA1 GENE IN pPICZαA AND TRANSFORMATION INTO

P. pastoris

The gene corresponding to ApoA1 was amplified using the primers as described in section 2.2.2. After amplification, the PCR product was cloned into a T-A cloning vector pXcmKn12, and subsequently restriction digested using EcoRI and NotI to sub-clone into the target vector pPICZαA. The resulting construct was transformed into competent E. coli DH5α cells and selected by resistance to 50µg/ml Zeocin. The construct was verified for correct integration of target gene by restriction digestion analysis and DNA sequencing using AOX1-specific primers. The sequencing data is analysed in Fig. 2.1, highlighting important features of the construct. The resulting construct, pPICZα-ApoA1 (Fig. 2.2) contained an upstream αmating factor signal sequence for secretion of the expressed recombinant protein followed by the gene of interest. It also contained a resistance gene to the antibiotic Zeocin, which would act as a selection marker upon transformation of this construct. An important feature to note is the incorporation of a stop codon in the reverse primer to exclude the optional c-myc epitope and hexahistidine tag present in the vector. This has been done in order to express the protein as close as possible to its native state, to maintain maximum activity. This also resulted in minimal inclusion of additional amino acids, thereby eliminating the need for additional digestion steps during downstream processing.

tcaaaaaacaactaattattcgaaacgatgagatttccttcaatttttactgctgtttta S K N N -L F E T M R F P S I F T A V L ttcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggca F A A S S A L A A P V N T T T E D E T A caaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgct Q I P A E A V I G Y S D L E G D F D V A gttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgcc V L P F S N S T N N G L L F I N T T I A agcattgctgctaaagaagaaggggtatctctcgagaaaagagaggctgaagctgaattc S I A A K E E G V S L E K R E A E A E F gatgaacccccccagagcccctgggatcgagtgaaggacctggccactgtgtacgtggat D E P P Q S P W D R V K D L A T V Y V D gtgctcaaagacagcggcagagactatgtgtcccagtttgaaggctccgccttgggaaaa V L K D S G R D Y V S Q F E G S A L G K cagctaaacctaaagctccttgacaactgggacagcgtgacctccaccttcagcaagctg Q L N L K L L D N W D S V T S T F S K L cgcgaacagctcggccctgtgacccaggagttctgggataacctggaaaaggagacagag R E Q L G P V T Q E F W D N L E K E T E ggcctgaggcaggagatgagcaaggatctggaggaggtgaaggccaaggtgcagccctac G L R Q E M S K D L E E V K A K V Q P Y ctggacgacttccagaagaagtggcaggaggagatggagctctaccgccagaaggtggag L D D F Q K K W Q E E M E L Y R Q K V E ccgctgcgcgcagagctccaagagggcgcgcgccagaagctgcacgagctgcaagagaag P L R A E L Q E G A R Q K L H E L Q E K ctgagcccactgggcgaggagatgcgcgaccgcgcgcgcgcccatgtggacgcgctgcgc L S P L G E E M R D R A R A H V D A L R acgcatctggccccctacagcgacgagctgcgccagcgcttggccgcgcgccttgaggct T H L A P Y S D E L R Q R L A A R L E A ctcaaggagaacggcggcgccagactggccgagtaccacgccaaggccaccgagcatctg L K E N G G A R L A E Y H A K A T E H L agcacgctcagcgagaaggccaagcccgcgctcgaggacctccgccaaggcctgctgccc S T L S E K A K P A L E D L R Q G L L P gtgctggagagcttcaaggtcagcttcctgagcgctctcgaggagtacactaagaagctc V L E S F K V S F L S A L E E Y T K K L aacacccagtgagcggccgccagctttctagaacaaaaactcatctcagaagaggatct g N T Q -

Signal sequence

Apolipoprotein A-I sequence Five of the clones that exhibited maximum resistance to Zeocin and verified to have the gene incorporated into the genome were further studied for preliminary expression in shake flasks.

EXPRESSION OF rhApoA1 IN P. pastoris

Five clones that exhibited high resistance to Zeocin (2 mg/ml) were independently inoculated into BMGY media for expression studies. Induction was carried out as explained in the methods section.

A B

As evident in figure 2.5(a), all screened clones expressed rhApoA1, and the intensity of expression increased with the duration of induction (Fig. 2.5(b)). The negative control (P. pastoris X-33 transformed with the pPICZαA vector) showed no expression of rhApoA1, as expected. Upon the conclusion of this preliminary study, the clone with highest rhApoA1 expression level (clone ID F1) was taken up for further scale-up studies. In the glycerol batch phase (first 24 hrs), there was a consistent drop in pO2 levels (red) until it reached set point (15%), subsequently, the agitation (blue)

and aeration (green) increased to maintain the pO2 levels. After exhaustion of substrate, the agitation decreased until set low-limit (200 rpm), in time for the commencement of induction with methanol (pink spikes). With every methanol addition episode (every 12 hrs), there was a corresponding increase in agitation indicating consistent metabolism. At the end of the production, the expression levels were verified by SDS-PAGE and western blotting (Fig. 2.6(b)) prior to A B induction (UI) and after 5-days of induction with 0.5% methanol (5d), and the expression in the bioreactor was successfully confirmed.

CONCLUSION

This chapter highlights the cloning of wild type ApoA1 gene in the expression vector pPICZαA and the successful expression of rhApoA1 in Pichia pastoris.

Reproducible and consistent expression levels were observed in shake-flasks cultures over two batches, paving way for preliminary data for developing a bioprocess for production of rhApoA1 in P. pastoris. Subsequently, the production was also reproduced under chemostat conditions (temperature, pH, pO2 levels maintained), and the expression was verifiable by standard biochemical methods (SDS-PAGE and Western Blotting).

A number of expression systems have been explored for the generation of recombinant ApoA1. The bacterial expression host E. coli has been previously demonstrated for rhApoA1 production [START_REF] Panagotopulos | Bacterial Expression and Characterization of Mature Apolipoprotein A-I[END_REF], but low expression levels even after codon optimisation [START_REF] Ryan | Optimized Bacterial Expression of Human Apolipoprotein A-I[END_REF] and intracellular expression have been limiting factors in scaling up production. P.

pastoris is an established system for the heterologous expression of proteins, in producing several of them at gram per litre levels [START_REF] Klein | High-Level Secretion of a Wheat Lipid Transfer Protein inPichia Pastoris[END_REF]. The ability of P.

pastoris to secrete proteins into the media helps reduce the complexity in downstream processing [START_REF] Brake | Alpha-Factor-Directed Synthesis and Secretion of Mature Foreign Proteins in Saccharomyces Cerevisiae[END_REF], especially when the target protein is being expressed in its near-native state without any additional affinity tags.

INTRODUCTION TO MIXED-MODE CHROMATOGRAPHY

Mixed-mode chromatography or multi-modal chromatography has been developed for the rational use of multiple interactions in a controlled manner, in contrast to non-specific interactions (Pezzini et al. 2015). Mixed-mode ligands have been in existence for a long time, with hydroxyapatite chromatography, a combination of ionic and metal-affinity interactions, possibly making it the oldest form of mixed-mode chromatography [START_REF] Gorbunoff | The Interaction of Proteins with Hydroxyapatite: III. Mechanism[END_REF]. Several other chromatographic methods, like dye-affinity, Histidine ligand affinity, peptide-affinity chromatography could also be classified as mixed-mode and have been well characterised [START_REF] Pezzini | A Study on the Nature of Interactions of Mixed-Mode Ligands HEA and PPA HyperCel Using Phenylglyoxal Modified Lysozyme[END_REF]. Several mixed-mode ligands are available in the market, and some of them have been listed below in table 3.1. In order to purify rhApoA1 for potential therapeutic application, it was necessary to device a purification process that would specifically capture rhApoA1 from the P. pastoris expression broth in minimal steps. In an attempt to reduce the number of processing steps, the ApoA1 gene was already cloned in a manner that would eliminate the optional hexahistidine tag present in the pPICZαA vector, so that the expressed protein (chapter 2) could be obtained in its near-native form, without the addition of any affinity tags.

Previous reports on the overexpression of rhApoA1 in bacteria have all utilized a hexahistidine tag [START_REF] Bergeron | Characterization of Human Apolipoprotein A-I Expressed in Escherichia Coli[END_REF][START_REF] Panagotopulos | Bacterial Expression and Characterization of Mature Apolipoprotein A-I[END_REF][START_REF] Ryan | Optimized Bacterial Expression of Human Apolipoprotein A-I[END_REF], which was avoided in this work. From other sources (plasma, P.

pastoris), a few methods have been documented that could prove useful for the capture of ApoA1 (plasma or recombinant) in its native-state [START_REF] Feng | High Yield and Secretion of Recombinant Human Apolipoprotein AI in Pichia Pastoris[END_REF][START_REF] Aurélio | Cloud Point Method Applied to the Apolipoprotein A-I Extraction from Human Plasma and Its Identification by Tandem Mass Spectrometry[END_REF]). These methods have also been investigated for the recovery of the expressed rhApoA1 from P. pastoris expression broth.

In this chapter, novel mixed-mode chromatography methods have been developed to recover rhApoA1 from P. pastoris expression broth. The defined mixed-mode ligands HEA HyperCel, PPA HyperCel (Pall Life Sciences) and CaptoMMC (GE Healthcare) have been evaluated for their ability to capture rhApoA1 from P. pastoris expression broth (highlighted in rows 1, 3 and 15, respectively). These mixed-mode sorbents have been extensively characterised in our laboratories [START_REF] Ranjini | Study of the Mechanism of Interaction of Antibody (IgG) on Two Mixed Mode Sorbents[END_REF][START_REF] Pezzini | A Study on the Nature of Interactions of Mixed-Mode Ligands HEA and PPA HyperCel Using Phenylglyoxal Modified Lysozyme[END_REF]Pezzini et al. 2015), and hence were explored conditions for the capture of rhApoA1 from the P. pastoris expression broth.

Mixed mode ligands offer multiple-types of interactions, such as ionic, hydrophobic hydrogen bonding, and the balance between these interactions allow new selectivity [START_REF] Zhao | Ligands for Mixed-Mode Protein Chromatography: Principles, Characteristics and Design[END_REF]. The salt tolerance afforded by the hydrophobic character reduces the need for sample dilution; and is the key reason for choosing mixed mode chromatography for purifying rhApoA1. Below 20mS/cm, the primary mode of interaction is electrostatic and the mixed mode ligands used in this study have cation exchanger groups allowing an extended pH binding range compared to traditional ion exchangers which permit protein adsorption only near the pI of the target protein.

In addition, the efficiency of these mixed-mode ligands in the efficient capture of rhApoA1 have been compared with earlier published methods as described in this section. were from GE Healthcare (Uppsala Sweden).

All buffers and other reagents were prepared using analytical grade chemicals from Sigma Aldrich (Bangalore, India).

EXPERIMENTAL CONDITIONS FOR DERIVED PURIFICATION METHODS

PURIFICATION OF rhApoA1 BY CLOUD-POINT EXTRACTION

A cloud-point extraction protocol was derived from the method previously published for recovering plasma ApoA1 (Marco Aurélio Zezzi Arruda et al. 2011).

For preliminary optimisation experiments, various concentrations of NaCl and

Triton X-114 were added to 2 ml of P. pastoris expression broth containing ApoA1 at 4°C. The mixture was vortexed and allowed to stand for 5 mins at room temperature, until it separated into a dense surfactant-poor and a light surfactant-rich phase. The solution was then centrifuged at 1780g for 10 minutes to clearly separate out the two phases, following which the surfactant rich phase was transferred to a fresh tube. The surfactant rich phase was precipitated with cold-acetone to eliminate the detergent and the surfactant poor phase was precipitated with TCA followed by cold-acetone washes to eliminate the salts. All fractions were analysed over SDS-PAGE analysis for the presence of rhApoA1, and proteins in the surfactant rich & poor phases were estimated by Bradford's assay and analysed by 12% SDS-PAGE for verifying partitioning. Subsequently, under optimised conditions, the strategy was scaled-up for a larger volume (50 ml) of P. pastoris broth.

PURIFICATION OF rhApoA1 BY COLD-ACETONE PRECIPITATION

Purification of rhApoA1 from P. pastoris expression broth was carried out as described by [START_REF] Feng | High Yield and Secretion of Recombinant Human Apolipoprotein AI in Pichia Pastoris[END_REF]. Subsequent to expression of rhApoA1, the P.

pastoris culture was centrifuged at 4,000 rpm for 10 mins to separate the cells from the supernatant. Cold acetone (-20°C) was added to 20% final concentration and incubated at -10°C for 5hrs. After the incubation period, the mixture was again centrifuged at 12,000 rpm for 20 mins after which the acetone concentration in the supernatant was raised to 40%. Subsequently, the mixture was again incubated at -20°C for 5hrs, after which the mixture was centrifuged again. The pH of the supernatant was adjusted to 5.8, after which cold acetone was again added to 60% and centrifuged. The pellet containing rhApoA1 was resuspended in phosphate buffer (pH 7.4), desalted using a HiTrap

Desalting column (i.d. 1.6 cm x 2.5 cm, GE Healthcare) to remove any residual salt, and subjected to ion exchange chromatography using quaternary amine (Source 15Q, i.d. 0.5 cm x 9.5 cm, GE Healthcare), in phosphate buffer at pH 7.4, and eluted using a salt gradient from 0 to 0.5 M NaCl, followed by a step to 1 M NaCl.

PURIFICATION OF rhApoA1 BY ION-EXCHANGE CHROMATOGRAPHY

As an improvisation of the published method in section 3.2.2.2 [START_REF] Feng | High Yield and Secretion of Recombinant Human Apolipoprotein AI in Pichia Pastoris[END_REF]), the P. pastoris expression broth containing rhApoA1 was desalted and directly subjected to ion exchange chromatography to evaluate if the cold-acetone precipitation step could be eliminated.

In the first step, the P. pastoris expression broth was desalted using a HiPrep 26/10 column (i.d. 2.6 cm x 10 cm, GE Healthcare) with 50mM Tris, pH 8.0 at a flow rate of 7 ml/min. A method was programmed to process 10 ml of P. pastoris expression broth in a batch, yielding a desalted fraction of 17 ml volume. Five batches were performed and the eluted fractions were pooled for the subsequent ion exchange experiment.

Following desalting, the pooled fractions (~70 ml) were injected on to a pH 8.0 equilibrated Source 15Q column (i.d. 0.5 cm x 9.5 cm, GE Healthcare). After collecting the non-retained fraction, the bound proteins were eluted in steps by using 100 mM, 200 mM and 1 M NaCl.

Finally, the fractions eluted with 200 mM NaCl were further subjected to reverse phase chromatography on a Source 15RPC column (i.d. 0.64 cm x 10 cm, GE Healthcare). The column was equilibrated water + 0.1% TFA (buffer A), and the following gradient was applied with acetonitrile + 0.085% TFA (buffer B): 0-15%B over 20CV; 15-25%B over 20CV; hold at 30%B for 5CV; hold at 80%B for 5CV; and hold at 0%B for 5CV. Peak fractions were collected and analysed by SDS-PAGE.

CHROMATOGRAPHIC CONDITIONS FOR NOVEL PURIFICATION METHODS USING MIXED-MODE CHROMATOGRAPHY

In addition to the above-explored published methods, several mixed-mode chromatography methods have been investigated and optimised for the recovery of rhApoA1 from P. pastoris expression broth.

PURIFICATION OF rhApoA1 BY HEA HYPERCEL

Prepacked PRC HEA Hypercel column (i.d. 0.8 cm x 10 cm) was first equilibrated with 50 mM Tris buffer, pH 7.4, 250 mM NaCl (binding buffer). Salt was added to give the buffer the same conductivity as the P. pastoris expression broth. The pH of the load was raised to 7.4 using 10 N KOH. After the column was equilibrated, the sample was injected, and non-retained fractions were collected. After the absorbance reached baseline the salt in the buffer was eliminated, through a linear gradient with 50 mM Tris, pH 7.4 over 20 column volumes. Elution was then carried out in steps using 50mM sodium acetate buffers at pHs 6.0, 5.0, 4.0 and 3.0. The eluted fractions were analysed by SDS-PAGE and western blots to verify the purification of rhApoA1.

PURIFICATION OF rhApoA1 BY PPA HYPERCEL

Pre-packed PRC PPA Hypercel column (i.d. 0.8 cm x 10 cm) was first equilibrated with 50 mM Tris buffer, pH 7.4, 250 mM NaCl (binding buffer). Salt was added to give the buffer the same conductivity as the P. pastoris expression broth. The pH of the load was raised to 7.4 using 10 N KOH. After the column was equilibrated, the sample was injected, and non-retained fractions were collected. After the absorbance reached baseline the salt in the buffer was eliminated, through a linear gradient with 50 mM Tris, pH 7.4 over 20 column volumes. Elution was then carried out in steps using 50mM sodium acetate buffers at pHs 6.0, 5.0, 4.0 and 3.0. The eluted fractions were analysed by SDS-PAGE and western blots to verify the purification of rhApoA1.

PURIFICATION OF rhApoA1 BY CAPTO MMC

The Capto MMC column was first equilibrated with 50 mM acetate buffer, pH 5.0, 250 mM NaCl (binding buffer). The pH of the P. pastoris expression broth at the end of the fermentation batch was adjusted to pH 5.0 and injected on to the column. The non-retained flowthrough fraction was collected, and once the absorbance reached baseline, the salt was eliminated over a linear gradient using 50mM acetate buffer, pH 5.0 over 20 column volumes. Elution was then carried out in steps using 50 mM phosphate buffer at pH 7.0 and 50 mM Tris buffer at pH 8.5. The eluted fractions were analysed by SDS-PAGE and western blots to verify the successful purification of the target protein (rhApoA1).

B A

clearly not suitable for subsequent scaling-up to larger volumes and potential industrial exploitation.

PURIFICATION OF rhApoA1 BY COLD-ACETONE PRECIPITATION

Based on [START_REF] Feng | High Yield and Secretion of Recombinant Human Apolipoprotein AI in Pichia Pastoris[END_REF], capture of rhApoA1 was attempted by cold-acetone precipitation, followed by an ion-exchange chromatography polishing step. This is the only existing purification report available for the recovery of rhApoA1 expressed in P. pastoris. After three precipitation steps with cold acetone (-10°C)

for 5 h each, followed by isoelectric precipitation and polishing by anion exchange chromatography, rhApoA1 was successfully recovered from the P.

pastoris expression broth (Fig. 3.3(b)). All fractions from various steps were analysed by 12% SDS-PAGE, which revealed the successful purification of rhApoA1 (Fig 3.4(d)). The purified fraction (RPC #2) was also verified by western blotting using polyclonal anti-ApoA1 antibody. The eluted fraction also showed aggregates of rhApoA1, consistent with previously published literature [START_REF] Vitello | Studies on Human Serum High Density Lipoproteins. Self-Association of Apolipoprotein A-I in Aqueous Solutions[END_REF]. This method successfully circumvented the acetone precipitation steps, and we were able to recover rhApoA1 with an overall recovery of 33%. However, long processing times in desalting and limited flow-rates and difficulties in reproducibility of the RPC experiment were limiting factors in scaling this purification scheme.

PURIFICATION OF rhApoA1 BY MIXED-MODE CHROMATOGRAPHY

Despite a number of functional purification methods that were evaluated and improvised in the previous section, each technique showed severe limitations in their suitability for scale-up. It was hence imperative to look for novel purification approaches for efficiently capturing rhApoA1 from the P. pastoris expression broth. In this section, three defined mixed-mode ligands, HEA HyperCel (Pall Life Sciences), PPA HyperCel (Pall Life Sciences) and CaptoMMC (HE Healthcare), have been evaluated for their ability to capture rhApoA1.

PURIFICATION OF rhApoA1 BY HEA HYPERCEL

The ligand HEA HyperCel (Hexylamine) has been well documented for the capture of several proteins [START_REF] Ranjini | Study of the Mechanism of Interaction of Antibody (IgG) on Two Mixed Mode Sorbents[END_REF][START_REF] Pezzini | A Study on the Nature of Interactions of Mixed-Mode Ligands HEA and PPA HyperCel Using Phenylglyoxal Modified Lysozyme[END_REF]. It is hypothesised that the nature of interaction between proteins and the HEA HyperCel ligand is a combination of hydrophobic and electrostatic. Equilibration In this study, a certain amount of salt (250 mM NaCl) was added to the equilibration buffer to generate the same conductivity as that of the P. pastoris expression broth, in an attempt to reduce the number of sample processing steps prior to injection. The pH of the P. pastoris broth containing rhApoA1 was raised to 7.4 and subsequently injected on to the column. After passing the sample, the non-retained proteins were allowed to flow through, after which the salt in the buffer was eliminated in a linear gradient. Upon 12% SDS-PAGE analysis, it was evident that the bound rhApoA1 was recovered at pH 4.0 (fraction 2a, indicated by the arrow). Some of the higher molecular weight proteins were loosely bound and were eluted at pH 5.0, whereas other proteins were more strongly bound to the ligand and eluted out at pH 3.0. The method was efficient in recovering rhApoA1 with an overall yield of 56.25% (Table 3.2), and was reproducible.

However, eluting rhApoA1 at pH 4.0 was a cause of concern. Recent studies have reported the propensity of ApoA1 to form amyloid like structures at pHs 4.0 and below [START_REF] Ramella | Human Apolipoprotein A-I Natural Variants: Molecular Mechanisms Underlying Amyloidogenic Propensity[END_REF]. It was hence imperative to minimise the exposure of rhApoA1 to very acidic pHs.

PURIFICATION OF rhApoA1 by PPA HYPERCEL

The PPA HyperCel ligand (Pall Life Sciences) is a phenyl propyl amine derivative (Table 3.1), and has been demonstrated in the efficient capture of a number of proteins over a wide pH range [START_REF] Ranjini | Study of the Mechanism of Interaction of Antibody (IgG) on Two Mixed Mode Sorbents[END_REF].

In this study, the binding and elution conditions tested for purifying rhApoA1 was similar to that with HEA HyperCel. The pH of the same was adjusted to 7.4

and loaded on to an equilibrated PPA HyperCel column. After binding, the salt was eliminated through a linear gradient and bound proteins were subsequently eluted in a step-wise manner by lowering the pH, yielding the chromatogram in being a predominantly hydrophobic protein (it is a lipid-binding protein) exhibits greater affinity to the phenyl-derivative (PPA) over the alkyl-derivative (HEA). Such comparisons have been extensively carried out in the past with other proteins [START_REF] Ranjini | Study of the Mechanism of Interaction of Antibody (IgG) on Two Mixed Mode Sorbents[END_REF][START_REF] Pezzini | A Study on the Nature of Interactions of Mixed-Mode Ligands HEA and PPA HyperCel Using Phenylglyoxal Modified Lysozyme[END_REF]Pezzini et al. 2015), and the present data is consistent with literature.

In this work, HEA and PPA HyperCel ligands have both been efficient in selectively capturing rhApoA1 from the P. pastoris expression broth, but have demonstrated other difficulties in handling of the purified proteins, namely the lowering of pH to elute the bound protein. One possible solution is to rapidly titrate the collected elution fractions with a concentrated stock of Tris buffer at pH 8.5 to raise the pH to near-neutral levels.

A B A

PURIFICATION OF rhApoA1 BY CAPTO MMC

The multimodal Capto™ MMC ligand supports binding of proteins through hydrophobic, ionic and thiophilic interactions. Being a weak cation exchanger, it was expected to function in an inverse manner to the earlier tested HEA Hypercel (Section 3.3.2.1.). The column was equilibrated as described in the experimental section (acetate buffer at pH 5.0 containing 250 mM NaCl), which was the same pH and conductivity as that of the P. pastoris expression broth.

Thus no sample pre-treatment was required prior to injection of the sample on to the column. This is especially an added advantage when designing a process of industrial relevance.

Following sample injection, the absorbance at 280nm was monitored until the signal reached baseline. The elimination of salt over a linear gradient didn't cause any proteins to unbind, in line with the expected salt-independent binding behaviour of Capto™ MMC. Subsequently, clear sharp peaks were observed when the pH was raised to 7.0 and 8.5 in steps (Fig. 3.8(a)). Any strongly bound proteins were then washed out with 0.5M NaOH (CIP). m.wt. Although this band did not provide a signal with anti-ApoA1 antibodies in western blots (possibly due to lower quantity), they were confirmed to be rhApoA1-dimers by mass spectrometric analyses (in-gel digestion; data not shown). To sum up, rhApoA1 was successfully purified to 84% purity using Capto™ MMC in a single step with 69% yield. The purification efficiencies of both purification methods have been summarized in Table 3.2.

Binding at mildly acidic pH (lower than the pI of rhApoA1) is consistent with earlier literature on the Capto™ MMC ligand. Being a salt-tolerant ligand, Capto™ MMC was able to retain the bound protein despite the elimination of conductivity. Subsequently, upon a rise in pH (resulting in a reversal of charges on the surface of the target protein rhApoA1), the protein desorbed and eluted out in a single sharp peak. This result was highly reproducible over several runs and was subsequently scaled-up to expanded bed mode (Chapter 4).

COMPARISON OF NOVEL PURIFICATION METHODS WITH PREVIOUSLY PUBLISHED METHODS

While previously published methods discussed in this chapter (Section 3.3.1)

show reasonable efficiency in recovering rhApoA1 from P. pastoris expression broth, their efficiency in recovering maximal rhApoA1 in minimal number of steps is not suitable for further scaling up. The use of Triton X-114 to extract the hydrophobic rhApoA1 poses serious difficulties in eliminating all traces of the detergent without compromising on protein activity; precipitation with cold acetone is not pragmatically feasible for industrial scale-up, especially with large quantities of acetone and long incubation times that are required; the improvised ion-exchange chromatography method required extensive desalting prior to treatment, which is again not practically scalable.

The defined mixed-mode ligands that have been tested in this work, show great promise in capturing rhApoA1 directly out of the P. pastoris expression broth with minimum number of processing steps. The relative efficiencies of all the methods that have been compared are summarised below in Table 3.2. From the comparative data above, it was evident that the mixed-mode ligands were more effective in recovering rhApoA1, with Capto MMC (row # 6) proving to be most efficient in capturing rhApoA1 in a single step.

MASS SPECTROMETRIC ANALYSIS OF PURIFIED rhApoA1

The elution fractions from the HEA HyperCel, PPA HyperCel and CaptoMMC chromatographic experiment were subjected to in-solution digestion as described in the methods section, followed by analysis in an Agilent G6540A MS/MS Q-TOF mass spectrometer. The eluted fractions were independently subjected to trypsinisation and MS/MS analysis. Upon analysing the results using Agilent MassHunter software, each of the analysed fractions showed at least 65% sequence coverage with that of rhApoA1, confirming that the purified protein was indeed rhApoA1. The sequence coverage and list of identified peptides with their scores are detailed in figure 3.9 and table 3.3 respectively. Although extensive sequence coverage was observed in the MS analysis (greater than 65%), there were no tryptic peptide matches in the last 60 amino acids at the C-terminal, which hinted towards a possible truncation in the expressed protein.

Furthermore, MALDI-TOF analysis (Fig. 3.10) of intact undigested purified rhApoA1 revealed a lower molecular mass observed (21.99 kDa) than the expected molecular mass of 28.3 kDa. Suspecting proteolytic activity by P. pastoris, the expression of the protein was also subsequently studied on a protease-deficient strain of P. pastoris (strain SMD1168) to compare the completeness of the proteins expressed by the two strains (Chapter 4).

CONCLUSION

In this chapter, a number of novel purification strategies have been elucidated

and experimented to recover rhApoA1 from P. pastoris expression broth, and have been compared with methods that have previously been published in

INTRODUCTION TO STRAINS OF P. pastoris

Pichia pastoris is a versatile microorganism that is being widely exploited for the overexpression of recombinant proteins. Different proteins demand different phenotypes and features in the host. Common P. pastoris strains used in the heterologous expression of proteins are listed below in Table 4.1. GlycoSwitch ® strain, Man5 N-linked oligosaccharide structures 8. SuperMan5 och1 -, his - GlycoSwitch ® strain, Man5 N-linked oligosaccharide structures (histidine auxotroph) X-33 is the primary wild type strain, which is most widely used for applications that don't involve any glycosylation. The GS115 strain is obtained by knocking out his4 gene, which enables a second level of selection on these strains in addition to the antibiotic resistance. KM71 strains generate Mut s phenotypes upon transformation with an AOX1-promoter containing vector, which is suitable for high-density fermentations for the production of recombinant proteins that aren't sensitive to proteolytic degradation. On the other hand, the SMD1168 strains are protease A knockouts, which enable the expression of proteins that are sensitive to proteolytic degradation. In addition, there are new strains based on the GlycoSwitch ® technology, which enable the expression of proteins with humanised-glycosylation patterns [START_REF] Jacobs | Engineering Complex-Type N-Glycosylation in Pichia Pastoris Using GlycoSwitch Technology[END_REF].

then added to the culture, and then centrifuged at 4ºC, 5000 rpm for 10 mins.

The pellet was resulspended in 20 ml of ice-cold 1 M sorbitol, and centrifuged at 4ºC, 5000 rpm for 10 mins. The pellet was again resuspended in ~100-200 µl of Sorbitol yielding approximately 500-600 µl of competent SMD-1168 cells.

Transformation of ApoA1 gene into competent SMD-1168 cells: 100 µl of competent SMD-1168 cells were taken in a fresh 1.5 ml microcentrifuge tube, to which approximately 1 µg of PmeI-linearised pPICZα-ApoA1 plasmid was added and were incubated in ice for 10 mins. The contents were then transferred to a sterile pre-chilled 0.2 mm electroporation cuvette, and pulsed using a Multiporator (Eppendorf) in the yeast module at 1500 V with a time constant (τ) of 5 ms. After the electroporation, 900 µl of ice-cold 1 M Sorbitol was added to the culture, and 200 µl was spread on YPD agar plates containing 100 µg/ml Zeocin.

Screening of positive transformants:

All colonies growing on the transformed plate were replica-plated on increasing concentrations of Zeocin up to 2 mg/ml.

The best growing clones were then selected for expression studies.

Verification of ApoA1 gene in the yeast genomic DNA:

The integration of the ApoA1 gene into the P. pastoris genome was verified by colony PCR analysis as described in Section 2.2.4. After the colony PCR run, the samples were subjected to 0.8% agarose gel electrophoresis and subsequently stained with Ethidium Bromide.

EXPRESSION OF rhApoA1 IN P. pastoris SMD-1168

The expression of rhApoA1 in SMD-1168 was carried out by a modified Mut s expression protocol. Briefly, a single colony was inoculated in 10ml of BMGY and was grown overnight at 30ºC, 220 rpm until the OD600 reached 10-11, and inoculated into 100 ml of BMGY to reach a starting OD600 of ~0.3. This culture was grown for 2 days at 30ºC, 250 rpm for biomass to gain and then centrifuged at 4ºC, 5000 rpm for 5 mins. The pellet was then weighed (x gms) and then resuspended in equal volume (x ml) of BMMY medium. Induction was then carried out with 1% methanol being replenished every 24 hrs supplemented with 0.1x YP. After the expression, the samples drawn at period intervals were analysed by 12% SDS-PAGE.

4.2.4. PURIFICATION OF rhApoA1 BY MIXED-MODE CHROMATOGRAPHY Purification rhApoA1 expressed in SMD-1168 was carried out similar to that optimised with rhApoA1 expressed in X-33. The Capto MMC column was first equilibrated with 50 mM acetate buffer, pH 5.0, 250 mM NaCl (binding buffer).

The pH of the P. pastoris expression broth at the end of induction was adjusted to pH 5.0 and injected on to the column (~10 ml of P. pastoris expression broth).

The non-retained flow through fraction was collected, and once the absorbance reached baseline, the salt was eliminated over a linear gradient using 50 mM acetate buffer, pH 5.0 over 20 column volumes. Elution was then carried out in steps using 50 mM phosphate buffer at pH 7.0 and 50 mM Tris buffer at pH 8.5.

The eluted fractions were analysed by SDS-PAGE and western blots to verify the successful purification of the target protein (rhApoA1). Subsequently, several colonies that exhibited resistance to Zeocin were screened with Zeocin concentrations of 500 µg/ml, 1 mg/ml and 2 mg/ml (Fig. After screening by colony PCR, one of the clones (#23) was taken further for expression studies (Section 4.3.2).

EXPRESSION OF rhApoA1 IN P. pastoris SMD-1168

Expression of rhApoA1 in SMD-1168 was carried out as described in methods section (Section 4.2.3). The OD600 of the cells were periodically checked for monitoring growth, and samples drawn every 24hrs were analysed over 12% The purification profile (Fig. 4.4(a)) was identical to that obtained earlier with rhApoA1 being purified from P. pastoris X-33. 12% SDS-PAGE analysis (Fig. 4.4(b)) again indicated towards no significant difference in molecular weight between rhApoA1 expressed in SMD-1168 and X-33 strains of P. pastoris and ApoA1 recovered from plasma (lane P).

COMPARISON OF rhApoA1 EXPRESSED IN X-33 AND SMD-1168

In order to unequivocally conclude on any potential truncation in rhApoA1 expressed in P. pastoris, the proteins expressed in different strains were compared by mass spectrometry.

Purified fractions containing rhApoA1 obtained from P. pastoris X-33 and SMD-1168 were analysed by 6540 UHD Q-TOF mass spectrometer (Agilent) and major species found in both samples were compared (Fig. 4.5). The major species in the purified rhApoA1 fraction from X-33 (22862.24 Da) was nearly identical to the major species in the purified rhApoA1 fraction from SMD-1168 (22869.30 Da).

This clearly showcased that that was no significant difference in molecular weight between the rhApoA1 proteins expressed in P. pastoris strains X-33 and SMD-1168.

A B

The MALDI-TOF profile of rhApoA1 from X-33 (Fig. 4.6(a)) showed a major species at 21995.8 Da, which was nearly identical to the major species reported earlier for plasma ApoA1 (22420.83 Da, Fig. 4.6(b)). Based on the above mass spectrometric comparisons, it was conclusive that there wasn't a significant difference in molecular mass between plasma and recombinant ApoA1 expressed in P. pastoris X-33 and SMD-1168 strains.

CONCLUSION

Truncation of recombinant proteins expressed in P. pastoris due to proteolytic activity has been reported earlier in literature (J. M. [START_REF] Cregg | Recombinant Protein Expression in Pichia Pastoris[END_REF]. In the present work, potential truncation was hypothesised owing to consistent lower molecular weight demonstrated by polyacrylamide gel electrophoresis (Chapter 3). Electrophoretic and mass spectrometric comparison of rhApoA1 expressed in X-33 with that expressed in SMD-1168 as well as plasma-derived ApoA1 clearly showcased no significance difference in molecular weight, indirectly confirming absence of any truncation in the expressed recombinant protein.

INTRODUCTION TO SCALE-UP OF INDUSTRIAL PROCESSES

Any bioprocess needs to be adequately scaled up to make it industrially viable. A typical bioprocess for the generation of recombinant therapeutics involves development of a stable strain expressing the target protein, followed by its production and subsequent purification. Scaling-up is typically carried out at both fermentation and downstream processing steps, with appropriate optimisation in order to maximise yield (Fig. 5.1). Various parameters need to be optimised during the process of scaling-up:

• Production/fermentation level: culturing conditions, feed rate, bioreactor and 5.0, and the corresponding increase in yield have been discussed.

SCALE-UP OF PURIFICATION SYSTEMS

Industrial downstream processing of therapeutic proteins typically involves a clarification or pretreatment step followed by a preparative capture chromatography purification step and further polishing to derive the final purified protein, prior to filtration and formulation [START_REF] Rathore | Scale-Up and Optimization in Preparative Chromatography: Principles and Biopharmaceutical Applications[END_REF]. Important factors that dictate the efficiency of a scaled-up purification process are listed below [START_REF] Garcia | Industrial Biochromatography: Engineering Aspects[END_REF]:

• Quality of gel material: affinity for the target protein, physical & chemical stability, non-biodegradability, inertness to the product, incompressibility

• Quality of sample to be chromatographed (free of particles that could clog columns)

• Reduction/Elimination of protease activity that could affect target protein

• Foaming due to tensioactive agents Sample pretreatment is especially important to ensure reproducibility of the process and reusability of the chromatographic resin. Alternate chromatographic approaches like fluidised beds or expanded beds could also be employed used for reducing the number of pretreatment, which in turn would reduce the composite particle containing an inert core material that is surrounded by an organic shell, enabling them to have a high sedimentation velocity at a reasonable particle size. Typically, by altering the particle composition, a Gaussian like distribution of particle size and particle density is achieved in an expanded bed.

Fluidisation of particles occurs when particles are pushed upwards in a column at a velocity corresponding to their sedimentation velocity. The degree of expansion of the bed is controlled by a number of factors: the size and density of the adsorbent beads, the linear flow velocity of the mobile phase, and the viscosity of the mobile phase. The bed is usually expanded to 2-3 times the packed-bed height. The absolute value for the degree of expansion depends on liquid density and viscosity. An increase in the viscosity of the buffer system causes an increase in the degree of expansion [START_REF] Mattiasson | 10 Physicochemical Basis of Expanded-Bed Adsorption for Protein Purification[END_REF]. Several recombinant proteins have been successfully purified by this method [START_REF] Chase | The Use of Affinity Adsorbents in Expanded Bed Adsorption[END_REF][START_REF] Hjorth | Expanded-Bed Adsorption in Industrial Bioprocessing: Recent Developments[END_REF][START_REF] Lamotte | Single Step Purification of a Wheat 9 kDa Lipid Transfer Protein Secreted by Pichia Pastoris[END_REF]. was further for scaled-up for production in a 5l benchtop bioreactor.

First, a single colony was inoculated and grown in baffled flasks containing 100ml of buffered glycerol-complex medium (BMGY, 100mM potassium phosphate buffer, pH 6.0, 13.4g/L YNB, 4x10 -4 g/L biotin, 10g/L glycerol and 150 µg/mL Zeocin™) up to an OD (600nm) of 4-8, and this was inoculated in 4L of BMGY in a BIOSTAT® Bplus laboratory 5l bioreactor with a starting OD600 of about 0.3. The temperature and pH were maintained at 30°C and 6.0 respectively, and dissolved oxygen level was maintained at 15% saturation by regulating aeration and agitation in a cascade manner. After complete consumption of glycerol in the medium (verified by standard glycerol assay; data not shown), a methanol fed-batch phase was initiated by adding methanol every 8hrs to a final concentration of 0.5%. Samples were drawn every 24hrs to follow the growth profile and protein production. After 120hrs of induction, the culture was harvested and directly chromatographed over an equilibrated Direct CST-1 column in Expanded Bed mode (Section 5.2.3). The expression of the recombinant protein was also monitored by SDS-PAGE and Western Blot analysis with samples drawn periodically (every 24hrs) from the bioreactor.

PURIFICATION OF rhApoA1 BY DIRECT CST-I IN EXPANDED-BED MODE

Once favourable conditions were achieved for the recovery of rhApoA1 using Capto™ MMC (Section 3.3.2.3), scale-up of purification was carried out in Expanded Bed mode using Direct CST-1 (GE Healthcare), which has the same chemistry as Capto™ MMC. The buffer conditions were as optimised in preliminary experiments. The column was equilibrated using 50mM acetate buffer, pH 5.0 and expanded by sequentially increasing flow-rate. The expression culture at the end of a fermentation batch (containing cells) was directly injected on to the expanded column to capture our target protein. Once binding and wash steps were completed, the column was allowed to settle and elution was carried out in downward flow using the same elution buffers. The various fractions were analysed by SDS-PAGE and western blots to verify the presence of rhApoA1.

As a polishing step, the elution fractions from the EBA chromatographic run were subjected to ion-exchange chromatography using Resource Q anionexchange column (GE Healthcare). The column was equilibrated with 20mM phosphate buffer, pH 7.0, after which the sample (fraction #2 from EBA experiment) was injected on to the column. After the absorbance at 280nm reached baseline, elution was carried out in steps containing 5%, 15% and 30% of 20mM phosphate buffer, pH 7.0, 1M NaCl. All collected fractions were analysed by 12% SDS-PAGE.

RESULTS

SCALED-UP EXPRESSION OF rhApoA1

Production of rhApoA1 in 5l benchtop bioreactor was carried out under conditions similar to that that were the maintained with the 2l benchtop bioreactor (Section 2.3.3). Briefly, an appropriate volume of preculture in BMGY containing 200µg/ml Zeocin was inoculated into a sterile bioreactor containing BMGY medium to a starting OD600 of 0.3. For the first 24hrs, cells were allowed to grow in the biomass accumulation phase in glycerol-containing medium (BMGY).

Once all glycerol was exhausted, the pH of the medium was lowered to 5.0, and the induction phase was commenced with methanol being fed every 8hrs to a final concentration of 0.5% (Fig. 5.2, turquoise curve). This ensured consistent replenishment of methanol and continued production of the target protein (rhApoA1). As monitored with the 2l bioreactor production batch, the pH and temperature were maintained in real time, using sterile concentrated reservoirs of acid/base and heating jacket respectively. From Fig. 5.3, it is evident that the cells were consistently metabolising the substrate supplied, inferable from the curves corresponding to pO2 levels (red) and agitation (blue). An increase in amount of base added to the bioreactor (yellow) is also an indication of secretion of proteins into the culture lowering the pH.

During the production, samples were drawn at various time points to check for glycerol consumption, target protein secretion, consistent growth and possible contamination. OD600 values of samples drawn at various time points revealed good biomass accumulation in the glycerol phase, and stable growth throughout the induction period (Fig. 5.4). Furthermore, microscopic examination revealed stable budding of yeast cells up to the 5 th day of production (Fig. 5.5) and the absence of any contamination.

After the completion of the production batch, samples drawn at various time points were analysed by SDS-PAGE, which revealed consistent expression of rhApoA1 (Fig. 5.6), as observed earlier with the 2l bioreactor run. The expression profiles of shake flask cultures were compared with bioreactor studies (Table 5.1), and the chemostat conditions clearly enabled the achievement of nearly twice the biomass (greater OD600) and protein yield (43.8 mg/l of production). The entire culture from the 4l production batch was utilised for one batch of purification in Expanded Bed Adsorption (Section 5.3.2).

PURIFICATION OF rhApoA1 BY DIRECT CST-I

In order to test the industrial applicability of this purification method, an Expanded Bed Adsorption resin: Direct CST I, was tested for its efficiency in capturing rhApoA1 from a crude feed containing P. pastoris cells directly from the bioreactor. The Direct CST I ligand has the exact same chemistry of Capto™ MMC, and has earlier been characterised using BSA and Myoglobin as model proteins [START_REF] Li | Expanded Bed Adsorption/desorption of Proteins with Streamline Direct CST I Adsorbent[END_REF].

Expanded bed adsorption enables the direct affinity capture of target proteins from crude samples. In our case, about 4 litres of unclarified P. pastoris culture (after a production batch in a benchtop bioreactor with 120hrs of induction, section 5.3.1) was injected to an expanded Direct CST I column. After the cell culture was completely passed through and the absorbance at 280nm reached baseline (washing completion), the flow was stopped and the column was allowed to settle. Subsequently, the salt was eliminated and pH was raised to elute bound proteins as earlier optimised in the packed column experiments with CaptoMMC (Section 3.3.2.3).

The chromatographic profile (Fig. 5.7a) was similar to that obtained with the packed 1ml HiTrap Capto™ MMC column (Fig. 3.8a). As expected, the peak eluted at pH 7.0 predominantly contained rhApoA1 (79% as quantified from the gel;

Fig. 5.7b

). There were still small amounts of other contaminating proteins (higher m.wt.), which needed to be eliminated.

An additional ion-exchange polishing step with Resource Q anionic exchanger column (GE Healthcare) was introduced. The polishing step was effective with 20mM sodium phosphate buffer (Fig. 5.7c), when rhApoA1 was eluted with just 50mM NaCl. At 300mM NaCl, other contaminating proteins co-eluted along with rhApoA1 and its dimer. Overall, about 136 mg of rhApoA1 was successfully purified in a single Direct CST I Expanded Bed Adsorption experiment from approximately 4l of unclarified expression culture to greater than 96% purity upon polishing with Resource Q.

The purification efficiencies of packed-column and expanded-bed modes were compared (Table 5.2), and the scale-up was shown to be effective.

The scale-up of purification of rhApoA1 using Direct CST-I in expanded bed adsorption mode was unequivocally established, and this paved way to the development of an integrated process (Section 5.3.3) for the production and rapid one-step capture of rhApoA1 expressed in P. pastoris.

DEVELOPMENT OF AN INTEGRATED PROCESS FOR THE PRODUCTION & PURIFICATION OF rhApoA1

For any production/purification process to be industrially viable, the focus is always on minimising the number of processing steps while maximising recovery/yield. In this study, the rhApoA1 has been expressed without the addition of any affinity tag so that it may be generated close to its native Cysteine residues in its amino acid sequence [START_REF] Brewer | The Amino Acid Sequence of Human Apoa-I, an Apolipoprotein Isolated from High Density Lipoproteins[END_REF] First, a single colony was inoculated and grown in baffled flasks containing 100ml of buffered glycerol-complex medium (BMGY, 100mM potassium phosphate buffer, pH 6.0, 13.4g/L YNB, 4x10 -4 g/L biotin, 10g/L glycerol and 150 µg/mL Zeocin™) up to an OD (600nm) of 4-8, and this was inoculated in 2L of BMGY in a BIOSTAT® Bplus laboratory 2l bioreactor with a starting OD600 of about 0.3. The temperature and pH were maintained at 30°C and 6.0 respectively, and dissolved oxygen level was maintained at 15% saturation by regulating aeration and agitation in a cascade manner. After complete consumption of glycerol in the medium (verified by standard glycerol assay; data not shown), a methanol fed-batch phase was initiated by adding methanol every 12hrs to a final concentration of 0.5%. Samples were drawn every 24hrs to follow the growth profile and protein production. Protein production was monitored by SDS-PAGE analysis. After 120hrs of induction, the culture was harvested and the cell-free broth was frozen at -80°C. scaled-up to be produced in a 2l benchtop bioreactor. The culturing conditions and parameters were similar to that optimised for the production of wild type rhApoA1, as explained in methods (Section 6.2.5). In both cases, the temperature and pH were maintained at 30ºC and 6.0, respectively. Dissolved oxygen levels were maintained at 15%, by a combination of agitation and aeration systems in a cascading manner. After the first 24hrs, and upon consumption of glycerol in the medium, induction was carried out by addition of 0.5% methanol every 12%.

Samples were drawn at periodic intervals for analysis by 12% SDS-PAGE. In the second approach (Fig. 6.10), the elimination of salt did not support the consistent binding of rhApoA1-Milano to the CaptoMMC resin, evidenced by the loss of certain amount of protein in the non-retained fraction (Fig. 6.10(b), Fraction 'FT'). As observed with the first approach, there was a small fraction of the target protein which bound stronger to the resin, evidenced by a faint signal in the elution fraction at pH 8.5 (Fig. 6.10, Fraction #2). While evaluating CaptoMMC for the capture of rhApoA1-Paris, two strategies were envisaged (similar to previous section). Binding was carried out at pH 4.5, with 250mM NaCl (moderate conductivity) in all cases. The following two elution strategies were evaluated: (i) rise in pH and subsequently conductivity; and (ii) elimination of salt followed by a rise in pH.

By the first strategy, all rhApoA1-Paris in the P. pastoris expression broth was captured by the ligand, however, with varying degrees of binding. This was substantiated by the presence of the bound rhApoA1-Paris in all elution fractions (Fig. 6.12(b)). Yet again, numerous other bands were present in each elution fraction demanding further polishing.

The alternate strategy of eliminating salt and subsequently rising the pH proved to be more effective in the recovery of rhApoA1-Paris from the P. pastoris expression broth. The bound protein was recovered in the elution step at pH 8.5, again indicating a stronger binding to CaptoMMC than wild type rhApoA1.

Despite the intriguing chromatographic profiles of rhApoA1-Paris on both HEA HyperCel and CaptoMMC, further polishing is required to recover the target protein in its pure form. achieved upon optimization of methanol induction phase. This production is significantly higher than that reported in CHO cells.

For any production/purification process to be industrially viable, the focus is always on minimising the number of processing steps while maximising recovery/yield. In this study, the rhApoA1 has been expressed without the addition of any affinity tag so that it may be generated close to its native conditions, in turn reducing the number of post-processing steps like proteolytic cleavage to remove tags, etc.

Eventually, a scalable two-step method has been developed: using mixed-mode chromatography to capture the expressed rhApoA1 in continuous mode without any pre-treatment or clarification followed by an anion exchange polishing step, which demonstrates greater advantages in terms of efficiency and number of processing steps in comparison to the previously published 12-step method with lower scalability [START_REF] Feng | High Yield and Secretion of Recombinant Human Apolipoprotein AI in Pichia Pastoris[END_REF]).

In addition, two naturally occurring variants of rhApoA1 (Milano and Paris) have been generated and produced in 2 litre benchtop bioreactors. Further optimisation and process development is required before scaling up to produce them as therapeutic proteins.

After the generation of the variants of rhApoA1, comprehensive comparative studies (biochemical and in vivo) would help understand better the mechanisms by which the Milano and Paris variants are more protective. In conclusion, this thesis has worked towards setting a foundation for further developmental work to be done in order to generate therapeutic ApoA1 molecules.

Figure R. 1 .

 1 Figure R.1. Le mécanisme de transport inverse de cholestérol

  (R173C) et Paris (R151C) R.2. CLONAGE ET EXPRESSION DE TYPE SAUVAGE DE L'ApoA1 HUMAINE RECOMBINANTE DANS P. pastoris X-33 R.2.1. MÉTHODES La séquence correspondant à l'ApoA1 a été amplifié en utilisant des amorces spécifiques sa séquence, et a été clone dans le vecteur pPICZαA et transformé en Pichia pastoris X-33 compétente, en suite les transformants ont été sélectionnés la résistance à la Zéocine™ (jusqu'à 2 mg / ml). Un étude préliminaire d'expression a été réalisée sur plusieurs clones de haute résistance sur les cultures en flasques, avec induction effectuée en utilisant 0,5% de méthanol chaque 24h pour 120hrs. Par la suite, le clone exprimant le plus élevé a été en outre pour l'expression dans un bioréacteur de 2L dans conditions régulées: Temp 30°C, pH 6,0, 15% saturation d'oxygène, induction avec 0,5% de méthanol chaque 12hrs. L'expression a été analysée par SDS-PAGE, dot-blot et Western blot. R.2.2. RÉSULTATS ET DISCUSSION Après le clonage, le construit (pPICZα-ApoA1) a été soumis à un séquençage d'ADN et a été vérifiée. La construit ete transforme par électroporation dans compétentes cellules de Pichia pastoris X-33, un certain nombre de transformants présentait une haute résistance à la zéocine et cinq d'entre eux ont été repris pour les études d'expression sur les cultures en flacons. Suite les études sur les cultures en flacons, un clone a été prise pour l'expression dans un bioréacteur. Une colonie unique a été inoculée et cultivée dans des flacons à déflecteurs de 100mL de milieu de glycérol-complexe (Buffered Complex Glycerol Medium, BMGY) jusqu'à ce que la DO (600 nm) atteint de 4 au 8, ce qui a été inoculé dans 2 litres d'BMGY dans un BIOSTAT® Bplus 2l bioréacteur. Les paramètres ont été maintenus constamment, et lors de la consommation complète du glycérol dans le milieu, une phase méthanol fedbatch a été initiée par l'addition de methanol toutes les 12 heures jusqu'à une concentration finale de 0,5% pour 120hrs. L'expression des protéines a été vérifiée par dot-blot, SDS-PAGE et analyse western blot.

Figure R. 2 .

 2 Figure R.2. Profil (A) et l'analyse SDS-PAGE et western blot (B) de l'expression de rhApoA1 expression in P. pastoris X-33.

  pastoris : HEA HyperCel (Pall Life Sciences) et Capto MMC (GE Healthcare), dont les structures sont à la Fig. R.3. Les conditions de chromatographiques ont été établies sur la base des directives de leur fabricant respectif, et les différentes fractions recueillies ont été analysées en utilisant SDS-PAGE et techniques Western blot.

Figure R. 3 .

 3 Figure R.3. Structures de ligands (A) HEA HyperCel et (B) Capto™ MMC Après avoir optimisé avec succès conditions chromatographiques pour récupérer rhApoA1, les protéines purifiées ont été digérés par la trypsine et analysées sur un-ESI-Q-TOF LC spectromètre de masse (modèle Agilent G6540A) pour évaluer la couverture de la séquence, afin de vérifier la protéine purifiée .

Figure R. 4 .

 4 Figure R.4. Purification de l'rhApoA1 a l'aide de l'HEA HyperCel : (A) Chromatogram et (B) analyse de SDS-PAGE 12%.

Figure R. 5 .

 5 Figure R.5. Purification de l'rhApoA1 a l'aide de Capto MMC : (A) Chromatogram, (B) analyse de SDS-PAGE 12% et (C) Blot western

Figure R. 6 .

 6 Figure R.6. Analyse spectrométrie en mass (ESI-Q-TOF) de fractions contenant la rhApoA1 purifiées à partir P. pastoris X-33 et SMD1168

R

  .6). Cela a conduit à croire que la protéine était en fait complète et intact.R.5. MISE A L'ECHELLE DE PRODUCTION ET DE PURIFICATION DErhApoA1 CHEZ P. pastoris X-33 R.5.1. MÉTHODES Suite a l'expression et la purification de rhApoA1 en échelle laboratoire, les études pour la mise à l'échelle de la production et la purification de rhApoA1 a été envisagé. La première étape vers l'intensification de l'expression a été effectuée dans un bioréacteur de 5l capacité. Les paramètres de l'expression ont été réglés comme ils l'étaient dans les lots de bioréacteurs 2l (voir section 2.1), et l'expression des protéines ont été analysées par SDS-PAGE et dot-blot.À la fin du lot de production, toute la culture de P. pastoris a été passé à travers une colonne équilibrée directe CST-I en mode "adsorption par lit expansé". Les conditions de tampon ont été maintenues comme il été optimisée dans des colonnes a l'echelle laboratoire, et le profil a été surveillée par l'absorbance à 280 nm. Les différentes fractions ont été recueillies et analysées par SDS-PAGE.R.5.2. RÉSULTATS ET DISCUSSIONLa profil de production de la rhApoA1 a ete suivre par SDS-PAGE et les niveaux de production de la rhApoA1 été comparables ont obtenus dans le bioréacteur de 2l (section R.2.2). Un taux d'addition de méthanol plus fréquente (chaque 8 heures) assurée l'induction suffisant de cellules en croissance. L'examen microscopique périodiques d'échantillons a vérifié l'absence de contamination dans la culture. La production en bioréacteur était très reproductible.

  Fig. R.7) en utilisant Direct CST-I (la même chimie de ligand comme Capto™ MMC), était similaire à celle obtenue dans des conditions à colonne paquée (Section R.3.2). Analyse par SDS-PAGE a confirmé la capture de tous rhApoA1 du milieu à une très haute concentration.

Figure R. 7 .

 7 Figure R.7. Purification de rhApoA1 par Direct CST-I en mode « adsorption lit expansee ». (A) Chromatogramme et (B) 12% SDS-PAGE de la charge (L), les fractions non retenues (FT) et éluée à pH 7,0 (fractions 1 -4) et 8,5 (fractions 5 -8). (C) 12% de l'analyse SDS-PAGE de fractions provenant Resource Q échangeuse d'ions de polissage étape: charge (L),

Figure

  Figure R.8. Analyse « Dot-Blot » de l'expression de l'ApoA1-Milano et l'ApoA1-Paris surexprimé chez P. pastoris X-33

  molecules are used to modulate the membrane fluidity of cells: by varying their concentration with changes in temperature. The structure of cholesterol has been elucidated in Fig. 1.1.

Figure 1 . 1 .

 11 Figure 1.1. Structure of Cholesterol (Reproduced from Wikipedia EN: http://en.wikipedia.org/wiki/Cholesterol)

Figure 1 . 2 .

 12 Figure 1.2. Lipoprotein structure highlighting components comprising hydrophilic surface (proteins, polar lipid head) and hydrophobic core (non-polar lipid, cholesterol, cholesterylester, triglycerides and fatty acids), reproduced from (Wasan et al. 2008).

  (i) Based on density/size (ii) Based on electrophoretic mobility (iii) Based on nature of Apo-protein content Among the above classification methods, classification of lipoproteins based on their density is most commonly adopted. The density of lipoproteins decreases with an increase in the ratio of lipids to proteins in them. Based on the density of the lipoprotein molecule, they are classified into high-density lipoproteins (HDL), low-density lipoproteins (LDL), intermediate-density lipoproteins (IDL), very low-density lipoproteins (VLDL) and chylomycrons. The relative sizes and densities of these various lipoproteins have been highlighted in Fig. 1.3.

Figure 1 . 3 .

 13 Figure 1.3. Classification of lipoproteins based on diameters (nm) and density (g/ml).

  : lipid-poor/lipid-free ApoA1 & discoidal HDL α-HDL: Spherical HDL particles (HDL2 & HDL3) γ-HDL: Large spherical particles with ApoE

Figure 1 . 4 .

 14 Figure 1.4. Schematic of Reverse Cholesterol Transport (RCT)

  first crystal structure of ApoA1 suggested a horseshoe like shape, constituted almost entirely of amphipathic α-helix that is punctuated by kinks at regular intervals introduced by proline residues[START_REF] Berg | Crystal Structure of Truncated Human Apolipoprotein A-I Suggests a Lipid-Bound Conformation[END_REF]). This amphipathic α-helix enables it to bind with lipids and cholesterol through its hydrophobic face and with the aqueous exterior with its polar face[START_REF] Murphy | High Density Lipoprotein: Assembly, Structure, Cargo, and Functions[END_REF]. A schematic ribbon structure of ApoA1 based on its crystal structure (PDB Accession Number 1AV1) is shown below in Fig. 1.5.

Figure 1 . 5 .

 15 Figure 1.5. Ribbon structure of ApoA1 (PDB accession # 1AV1).

  yet not conclusive data to showcase the behaviour of ApoA1 variants, especially Milano & Paris, under oxidative stress. It is hence of extreme interest to generate robust processes for the production of both wild type and cysteine variants of ApoA. 1.3.6. CLINICAL APPLICATIONS OF APOA1 -DIAGNOSTIC AND THERAPEUTIC Recent studies, based on the sensitivity of ApoA1 to MPO-mediated modification, have established oxidised ApoA1 as a diagnostic target for the early detection of atherosclerosis. Antibodies specific to oxidised residues on the surface of ApoA1 are currently being explored for use in the early diagnosis of atherosclerosis (Nakano and Nagata 2003).

  1.4. EXPRESSION HOST 1.4.1. INTRODUCTION TO EXPRESSION SYSTEMS The advent of recombinant DNA (rDNA) technology enabled the industrial production of heterologous proteins, making affordable therapeutics and reducing the cost of several diagnostic and therapeutic interventions. Human

  1.4.5. HETEROLOGOUS EXPRESSION OF PROTEINS IN P. pastoris: THE AOX1 SYSTEMPichia pastoris is one of approximately a dozen yeast species representing four different genera capable of metabolizing methanol. The methanol metabolic pathway appears to be the same in all yeasts and involves a unique set of pathway enzymes. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde in the peroxisome, generating hydrogen peroxide in the process, by the enzyme alcohol oxidase (AOX). The H2O2 produced by the AOX reaction is metabolised by catalase in the peroxisome (Fig.1.6). AOX is strongly repressed by many alternate carbon sources such as glucose, glycerol and even ethanol, and is induced by carbon starvation when grown with methanol as the sole carbon source. Although alcohol oxidase is not specific for methanol and is capable of oxidizing other primary alcohols, the activity of the enzyme decreases as the number of carbons in the alcohol increases. The conversion of methanol to formaldehyde is the rate-limiting step in utilizing methanol, due to the poor affinity of AOX to O2, and is regulated by increasing the amount of AOX enzyme in the cells[START_REF] Daly | Expression of Heterologous Proteins in Pichia Pastoris: A Useful Experimental Tool in Protein Engineering and Production[END_REF].

Figure 1 . 6 .

 16 Figure 1.6. Alcohol oxidase system.

  promotor region. This homologous recombination could result in either the conservation of native AOX1 activity or disruption of AOX1 gene, resulting in Mut + and Mut S phenotypes, respectively (Fig.1.7).

Figure 1 .

 1 Figure 1.7(a) Generation of Pichia pastoris transformants of Mut + phenotype.

Figure 1 .

 1 Figure 1.7(b) Generation of Pichia pastoris transformants of Mut S phenotype.

Figure 2 . 1 .

 21 Figure 2.1. DNA sequencing results of pPICZα-ApoA1 highlighting the α-mating factor signal sequence, cloning sites (EcoRI and NotI) and Apolipoprotein A-I gene.

Figure 2 .

 2 Figure 2.4. Colony PCR analysis of control (H2O), plasmids of vector (pZα-H6E) and vector containing ApoA1 gene (pZα-ApoA1), and P. pastoris transformed with vector (Pichia Zα-H6E) and P. pastoris clones from the YPD-agar containing 2 mg/ml Zeocin (from Fig. 2.3(b)). M7 ladder (SmartLetter MW-1700-10) was from Eurogentec (France).

Figure 2 . 5 .

 25 Figure 2.5. Comparative rhApoA1 expression analysis by SDS-PAGE of (a) all clones with control upon harvesting (stained with CBB); and (b) day-wise expression profiles of clones F1 and control (stained by Silver staining).

  2.3.3. EXPRESSION OF rhApoA1 IN P. pastoris (2 L BIOREACTOR)After preliminary studies on the expression of rhApoA1 in shake flasks, production was carried out in a 2 l benchtop bioreactor. The parameters were maintained as explained in Section 2.2.6, yielding the profile in Fig.2.6. The temperature and pH were maintained in real time by various controllers, and cell-growth and substrate metabolism was implied out of the dissolved oxygen levels maintained by aeration and agitation systems.

Figure 2 .

 2 Figure 2.6. (a) Profile and (b) SDS-PAGE & Western Blotting analysis of rhApoA1 expression in P. pastoris X-33.

  were performed on AKTA chromatographic platforms (AKTA Purifier, AKTA Explorer, AKTA FPLC, GE Healthcare) and operated by Unicorn software (GE Healthcare). Prepacked PRC HEA Hypercel and PRC PPA Hypercel columns were from Pall Life Sciences (France), and prepacked HiTrap Capto MMC, HiPrep 26/10, Source 15Q and Resource 15RPC

3. 2

 2 .4. VALIDATION OF PURIFIED rhApoA1: MASS SPECTROMETRY Structural characterisation of the purified rhApoA1 was carried out over tandem mass spectrometry using an Agilent G6540A LC ESI-Q-TOF MS/MS. The purified elution fractions were individually lyophilized and reconstituted in 100µl of 6 M Urea, 50 mM Tris-HCl, pH 8.0. The proteins were reduced by adding 5 µl of 200 mM DTT in Tris, pH 8.0 and incubating the mixture at room temperature for 1 hour, and subsequently alkylated by adding 20 µl of 200 mM Iodoacetamide and incubating at room temperature (dark) for 1 hour. After alkylation, the protein mixture was diluted with Tris, and incubated with Trypsin to a final w/w ratio of 1:50 (trypsin:protein) and incubated at 37°C for 16-20 hrs. After trypsinisation, the pH was reduced with formic acid and injected into the Agilent G6540A LC ESI-Q-TOF MS/MS. The data obtained were analysed using the Agilent Mass Hunter software and matched with the ApoA1 sequence.

  OF rhApoA1 BY CLOUD POINT EXTRACTION Aliquots of P. pastoris expression broth (2 ml each) were treated with various concentrations of Triton X-114 (2% to 20%) and NaCl (0% to 15%). After incubation at room temperature and centrifugation, the upper surfactant rich phase (~ 200 µl) and the lower surfactant poor phase (~1800 µl) were separated and quantified for their protein content. The partition coefficient was plotted as a function of concentration of Triton X-114 added for various concentrations of salt (Fig. 3.1(a)), and all fractions were analysed by 12% SDS-PAGE analysis (Fig. 3.1(b)).

Figure 3 . 1 .

 31 Figure 3.1. Cloud Point Extraction of rhApoA1 from P. pastoris expression broth under various [Triton X-114] & [NaCl]: (a) partition coefficient and (b) 12% SDS-PAGE.

Figure

  Figure 3.3. Purification of rhApoA1 by cold-acetone precipitation followed by ion exchange chromatography: (a) schematic of the method followed, (b) chromatogram of IEX over Source 15Q column and (c) SDS-PAGE analysis of various fractions.

  is typically under certain amount of conductivity to promote hydrophobic attraction(Fig 3.5). A combination of salt and pH is crucial for reducing this hydrophobic attraction and promoting electrostatic repulsion to elute the bound protein(Pezzini et al. 2015).

Figure 3 . 5 .

 35 Figure 3.5. Hypothetical mechanism of interaction of target protein with HEA HyperCel ligand. Binding is promoted by hydrophobic attraction under relatively higher pH and salt concentrations. Subsequently, the bound proteins are eluted by lowering the pH and salt concentrations which in turn promotes electrostatic repulsion.

  Finally, elution was achieved by lowering the pH sequentially (Fig 3.6(a)).

Figure 3 . 6 .

 36 Figure 3.6. Purification of rhApoA1 by HEA HyperCel. (A) Chromatogram and (B) 12% SDS-PAGE analysis of peak fractions.

Fig

  Fig. 3.7(a).

Figure 3 . 8 .

 38 Figure 3.8. Purification of rhApoA1 by Capto MMC. (A) Chromatogram, (B) 12% SDS-PAGE analysis of peak fractions and (C) Western blot analysis of eluted fraction at pH 7.0.

Figure

  Figure 3.9. Sequence coverage of rhApoA1 identified by trypsinisation followed by ESI-MS/MS

Figure 3 .

 3 Figure 3.10. MALDI-TOF spectrum of intact purified rhApoA1 from P. pastoris X-33

4. 2

 2 .5. COMPARISON OF rhApoA1 EXPRESSED IN VARIOUS P. pastoris STRAINS The intact masses of rhApoA1 expressed by the two different strains of P. pastoris (X-33 and SMD-1168) were compared by SDS-PAGE as well as mass spectrometry. Purified rhApoA1 fractions were measured by Agilent 6540 UHD Q-TOF mass spectrometer and analysed by Agilent Mass Hunter software to deconvolute and obtain intact mass of the measured fragments.4.3. RESULTS4.3.1. TRANSFORMATION OF pPICZα-ApoA1 AND SELECTION OF RESISTANTCLONESThe construct pPICZα-ApoA1 was linearised with PmeI and transformed into competent SMD-1168 strains as described in the methods section (Section 4.2.1).

Figure 4 . 1 .

 41 Figure 4.1. Replica plating of SMD-1168 cells transformed with ApoA1 gene at various concentrations of Zeocin

Figure

  Figure 4.2. Colony-PCR analysis of cell control (CC), vector control (VC), and five SMD-1168-ApoA1 clones (#12, #16, #23, #27, #48).

Figure

  Figure 4.4. Purification of rhApoA1 expressed in P. pastoris SMD-1168 by CaptoMMC. (A) Chromatogram and (B) 12% SDS-PAGE analysis of load (L), nonretained (FT), and elution fractions at pHs 7.0 & 8.5. Plasma derived ApoA1 (P) and rhApoA1 expressed in P. pastoris X-33 (Ref) were added as controls for comparing m.wt.

Figure 5 . 1 .

 51 Figure 5.1. Development of biopharmaceutical product (Thiry and Cingolani 2002)

size•

  Purification level: sample pretreatment, chromatography characteristics5.1.1. SCALE-UP OF EXPRESSION OF RECOMBINANT PROTEINSA vast majority of industrial processes using genetically modified microorganisms to overexpress recombinant proteins use one of the three species: Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris[START_REF] Thiry | Optimizing Scale-up Fermentation Processes[END_REF]. All three microorganisms showcase their relative advantages in terms of production levels, post-translational modifications, secretion capabilities, etc. In this work, the methylotrophic yeast Pichia pastoris has been exploited for the expression of rhApoA1.During scale-up, the primary aim is in increasing the size and volume of expression, in addition to optimising culture conditions to maximise yield of the heterologous protein. Temperature, pH and dissolved oxygen are parameters that are typically optimised during expression and scale-up of production of recombinant proteins[START_REF] Lerner-Marmarosh | Large Scale Purification of Detergent-Soluble P-Glycoprotein fromPichia Pastoris Cells and Characterization of Nucleotide Binding Properties of Wild-Type, Walker A, and Walker B Mutant Proteins[END_REF]. A reduction in induction temperature and pH conditions helps enhance the amount of recombinant protein produced by reducing protease activity. In the present study, the temperature of induction and pH of the medium have been optimised to 28°C

  expression studies in benchtop bioreactors were carried out on Sartorius-Stedim Biostat B-plus bioreactors (5l) monitored by MCSF Win data acquisition software (Sartorius). The Expanded Bed Adsorption experiments were carried out using Direct CST-1 resin (GE Healthcare, Uppsala, Sweden) loaded on a Streamline 25 housing (i.d. 2.5cm x 100cm, GE Healthcare, Uppsala, Sweden) controlled and monitored by Unicorn software (GE Healthcare, Uppsala, Sweden). 5.2.2. SCALE-UP OF PRODUCTION OF rhApoA1 TO 5L BENCH-TOP BIOREACTOR After initial batches of bioreactor studies on 2l benchtop reactors, the clone F1

Figure 5 . 3 .

 53 Figure 5.3. Expression profile of rhApoA1 production in 5l benchtop bioreactor.

Figure 5 . 4 .

 54 Figure 5.4. Optical Density (600nm) profile of samples drawn at various time points.

Figure

  Figure 5.5. Microscopic examination of budding yeast cells (transformed P. pastoris X-33) at 40x magnification.

Figure 5

 5 Figure 5.6. SDS-PAGE analysis of non-induced (UI) and samples drawn at 72h, 96h, and 120h of induction (3d, 4d and 5d, respectively). Band corresponding to rhApoA1 indicated by arrow.

Figure

  Figure 5.7. Purification of rhApoA1 by Direct CST-1 in Expanded Bed Adsorption mode. (A) Chromatogram and 12% SDS-PAGE of load (L), non-retained (FT) and eluted fractions at pHs 7.0 (fractions 1 -4) and 8.5 (fractions 5 -8). (B) Chromatogram and 12% SDS-PAGE analysis of fractions from Resource Q ionexchange polishing step: load (L), non-retained (FT), and eluted fractions at 5%, 15%, 30% and 100% of elution buffer (20mM phosphate buffer, pH 7.0, 1M NaCl).

  conditions. The application of Expanded Bed Adsorption sorbent Direct CST-I enables the development of an integrated process (Fig. 5.8) to directly capture the expressed rhApoA1 protein straight out of the bioreactor without any pretreatment or clarification.

Figure 5 . 8 .

 58 Figure 5.8. Integrated process for the direct purification of rhApoA1 from bioreactor using Direct CST-I in Expanded Bed Adsorption mode.

Figure 6 . 1 .

 61 Figure 6.1. Naturally occurring ApoA1 missense mutations or in-frame deletions classified according to their pathophysiological outcomes.

6. 2 . 2 .

 22 SITE-DIRECTED MUTAGENESIS OF ApoA1 GENE TO GENERATE MILANO & PARIS CONSTRUCTS Milano (R173C) and Paris (R151C) mutations were introduced by using the following sets of primers: Milano-F: 5' ACGAGCTGCGCCAGTGCTTGGCCGC 3' Milano-R: 5' GCGGCCAAGCACTGGCGCAGCTCGT 3' out for a total of 5 days; with methanol being replenished every 24hrs at 0.5%. At the end of the induction, comparative day-wise expression of select clones along with the control were analysed by dot-blot analysis with ApoA1-specific polyclonal antibodies. 6.2.5. SCALE-UP OF EXPRESSION IN 2L BENCHTOP BIOREACTOR One clone each from the pool of rhApoA1-Milano and rhApoA1-Paris were further subjected to expression studies in a 2l benchtop bioreactor.

6. 2

 2 .6. PRELIMINARY PURIFICATION OF rhApoA1-Milano & rhApoA1-Paris Purification of rhApoA1-Milano and rhApoA1-Paris were evaluated on two mixed-mode resins: HEA HyperCel (Pall Lifesciences) and CaptoMMC (GE Healthcare). The experimental conditions were similar to that optimised for the purification of wild type rhApoA1 (Section 3.3.2). 6.3.3. SCALE-UP OF EXPRESSION IN 2L BENCHTOP BIOREACTOR Based on the expression profiles of the three Milano & three Paris cultures in shake flask experiments, one clone each (Milano: E4 & Paris: E1) were further

Figure 6 .

 6 Figure 6.6. (a) Expression profile and (b) SDS-PAGE analysis of rhApoA1-Milano clone E4 on 2l benchtop bioreactor.

Figure

  Figure 6.7. (a) Expression profile and (b) SDS-PAGE analysis of rhApoA1-Paris clone E1 on 2l benchtop bioreactor.

Figure 6 . 8 .

 68 Figure 6.8. Purification of rhApoA1-Milano by HEA HyperCel. (a) Chromatogram and (b) 12% SDS-PAGE analysis of peak fractions

Figure 6 .

 6 Figure 6.10. Purification of rhApoA1-Milano by CaptoMMC. (a) Chromatogram and (b) 12% SDS-PAGE analysis of load (L), non-retained (FT), and peak fractions at pH 7.0 (#1), pH 8.5 (#2) & NaOH wash (#3).

  The differences in binding patterns of rhApoA1-Milano vis-à-vis wild type rhApoA1 to both HEA HyperCel and CaptoMMC resins strongly hint towards an increased hydrophobicity of rhApoA1-Milano. The stronger binding in HEA HyperCel, and the salt-dependent binding with CaptoMMC are both indicative of hydrophobic interactions, of an increased strength in comparison to the wild type rhApoA1 protein. A second factor to note is the presence of two populations of rhApoA1-Milano, one exhibiting weak interactions with CaptoMMC and the other of relatively stronger nature. It would be of interest to compare the functional activities of both fractions of rhApoA1-Milano to better understand their folding-related functional changes. Nevertheless, further polishing is required to recover the rhApoA1-Milano protein in its purified form for further analysis.6.3.5. BINDING PATTERNS OF rhApoA1-ParisThe binding profile of rhApoA1-Paris was evaluated on both HEA HyperCel and CaptoMMC chromatographic ligands.

Figure 6 .

 6 Figure 6.11. Purification of rhApoA1-Paris by HEA HyperCel. (a) Chromatogram and (b) 12% SDS-PAGE analysis of load, non-retained and peak fractions.

Figure

  Figure 6.12. Purification of rhApoA1-Paris by CaptoMMC. (a) Chromatogram and (b) 12% SDS-PAGE analysis of load (L), non-retained (FT), and peak fractions at pH 7.0 + 250mM NaCl (#1), pH 7.0 + 2M NaCl (#2) & NaOH wash (#3).

Figure

  Figure 6.13. Purification of rhApoA1-Paris by CaptoMMC. (a) Chromatogram and (b) 12% SDS-PAGE analysis of load (L), non-retained (FT), and peak fractions at pH 5.0 (#1), pH 7.0 (#2), pH 8.5 (#3) & NaOH wash (#4).

Table de Matières

 de / Table of Contents

	R.1. INTRODUCTION
	R.1.1. L'INTRODUCTION GÉNÉRALE Les troubles métaboliques, notamment le diabète, l'athérosclérose et les Résumé de thèse en français
	maladies cardiovasculaires sont les principales causes de morbidité et de
	mortalité dans les pays développés et en développement. L'obésité et la
	dyslipidémie sont les principaux facteurs de risque particulier dans les maladies
	cardiovasculaires
	L'expression de type sauvage et des
	variantes de l'Apolipoprotéine A-I
	humaine chez Pichia pastoris

Tableau R.1. Comparaison des méthodes de purification Méthode de purification Nombres d'étapes Rendement Purité d'rhApoA1 Reference

	Il était évident que les deux méthodes de purification développées dans cette
	étude ont été mieux par rapport aux méthodes déjà publiées.
	R.3.2.4.	IDENTIFICATION DE L'ApoA1 PURIFIÉE PAR SPECTROMÉTRIE DE
	A Suite de la production, l'rhApoA1 exprimé de P. pastoris SMD1168 a été purifié B C MASSE A La rhApoA1 purifié par les deux procédés de purification ont été digérés avec par chromatographie en mode mixte avec de ligand Capto™ MMC. Le procédé de
	trypsine et analysée par spectrométrie de mass sur ESI-Q-TOF MS/MS (Agilent). purification était similaire que la procédé developpe pour la rhApoA1 exprimé
	La rhApoA1 purifié par des deux procédés montré couverture de séquence sur le P. pastoris type sauvage, et les protéines purifiées à partir de APOA1 deux
	substantielle (~ 65%) avec la séquence d'ApoA1 humaine disponible sur NCBI. souches ont été comparées pour leur taille et leur séquence.
	1. Extraction par				(Marco Aurélio
	« Cloud-point » avec	2	55.97%	57.1%	Zezzi Arruda et al.,
	Triton X-114				2011)
	2. Précipitation avec acétone froid	14	60.00%	71.9%	(Feng et al., 2006)
	3. Chromatographie
	par mode mixte	1	56.25%	70.2%	Présente travail
	(HEA HyperCel)
	4. Chromatographie
	par mode mixte	1	52.50%	76.3%	Présente travail
	(PPA HyperCel)
	5. Chromatographie
	par mode mixte	1	68.89%	84.0%	Présente travail
	(CaptoMMC)

Ce fut une validation globale des méthodes de purification utilisées. Bien que la protéine purifiée a été vérifié pour être ApoA1, le poids moléculaire sur des gels SDS-PAGE a été constamment inférieur (~ 25-26kDa) que le poids moléculaire attendue (28 kDa).

Cela a conduit à la spéculation sur une éventuelle troncature de la protéine due à l'activité de la protéase de P. pastoris. Pour vérifier cette hypothese, le gène de l'ApoA1 a ensuite été transformé en une souche déficiente de la protéase de P. pastoris (SMD1168) et a induit avec du méthanol pour exprimer rhApoA1. Les détails expérimentaux et les résultats sont discutés en détail dans la section 4. R.4. CLONAGE, L'EXPRESSION ET LA PURIFICATION DE rhApoA1 CHEZ P. pastoris SOUCHE SMD-1168 (DEFICIENTE EN PROTEASE) Le poids moléculaire de rhApoA1 obtenu lors de l'expression de type sauvage souche P. pastoris X-33 a été constamment inférieur au poids moléculaire attendu, au cours des analyses SDS-PAGE. Nous regardé si il y avait une activité de protéase conduisant à cette troncature hypothétique, et essayé à exprimer la rhApoA1 dans une souche déficiente en protéase de P. pastoris : SMD1168. Les protéines exprimées étaient comparées entre les deux souches, de conclure globalement si la protéine a en effet été tronquée. R.4.1. MÉTHODES Compétente P. pastoris SMD1168 cellules ont été préparés et la construit pPICZα-ApoA1 a été transformé. Les transformants présentant une forte résistance à la Zéocine ont été choisis pour analyse de l'expression. L'expression a été effectuée d'une manière similaire à celle de la souche de type sauvage (X-33). Les niveaux d'expression ont été analysés par SDS-PAGE. R.4.2. RÉSULTATS ET DISCUSSION L'expression de rhApoA1 chez P. pastoris SMD1168 a été vérifiée par SDS-PAGE. Aucune différence significative du poids moléculaire été observée entre la rhApoA1 exprimée par X-33 et SMD-1168.

Tableau R.2. Comparaison des échelles de l'expression de la rhApoA1

	Paramètre\Échelle	Flasque	Bioréacteur 2L	Bioréacteur 5L
	Volume de culture	150 mL	1800 mL	4000 mL
	DO600nm finale	27.56	41.34	44.71
	pH d'induction	6,0	5,0	5,0
	Système de	Phosphate	phosphate	phosphate
	tampon		+ acide/base	+ acide/base
	Température de	30°C	28°C	28°C
	l'induction
	Rendement de la	22,4 mg/l	37,5 mg/l	43,8 mg/l
	rhApoA1

Le profil chromatographique de la purification (

Table 1 .3. HDL proteins and their major functions

 1

	S.No.	Protein	Function	Reference
	1. Apolipoprotein A-I	Main structural protein, lipid	(Borhani et al.
		(ApoA1)	binding, ABCA1 binding for	1997)
			cholesterol efflux, LCAT
			activation
	2. Apolipoprotein A-II	Enhances hepatic lipase
		(ApoA2)	activity
	3. Apolipoprotein D	Associated with LCAT,
		(ApoD)	progesterone binding
	4. Apolipoprotein E	LDL-receptor binding	(Barbier et al.
		(ApoE)		2006, 2)
	5. Apolipoprotein M	Transport of Sphingosine-1
		(ApoM)	Phosphate
	6. Serum Paraoxonase	Anti-oxidant and anti-	(Heinecke
		1 (PON1)	inflammatory effects	2010)

Table 1 .4. Variants of ApoA1

 1

	S.No.	ApoA1	Mutation	Physiological	Reference
		Variant		Consequence
	1. Milano	R173C	Reduced HDL but	(Weisgraber et
				healthy	al. 1983)
				cardiovascular
				system
	2. Paris	R151C	Reduced HDL but	(Bruckert et al.
				healthy	1997)
				cardiovascular
				system
	3. Pisa	L141R	Absence of HDL	(Miccoli et al.
				cholesterol	1996)
	4. Finland	K159R	Hypoalphalipo-	(Miettinen et al.
				proteinemia	1997)
	5. Iowa	G026R	Amyloidosis	(Benson et al.
					1991)
	6. Helsinky	K107Δ	Amyloidosis	(Ramella et al.
					2012)
	7. Fukuoka	E110K		(Takada et al.
					1990)
	Of the above listed variants, two variants, Milano and Paris, are characterised by
	a single point variation of Arginine to Cysteine at 173 and 151 respectively (Klon

Table 1 .5. MPO-mediated modifications of ApoA1 and its consequences S.No Type of Modification Modified amino acid generated

 1

			Functional
	Residues		Consequences
	modified	Mechanism	in vitro	Reference

Table 3 .1. Ligands for mixed-mode chromatography (Zhao, Dong, and Sun 2009). The ligands evaluated in this study have been highlighted in blue. S.No. Name Structure pKa Positively charged ligands

 3

Table 3 .2. Comparison of purification efficiencies of the various tested methods

 3

	S.	Purification Method	Number	Yield	rhApoA1	Reference
	No.	Employed	of Steps		purity*
	1. Cloud-point extraction				(Marco Aurélio
		with Triton X-114	2	55.97%	57.1%	Zezzi Arruda et al.
						2011)
	2. Cold-acetone precipitation	14	60.00%	71.9%	(Feng et al. 2006)
	3. Ion-exchange chromatography	3	32.45%	42.6%	Present work
	4. Mixed-mode
		chromatogr. (HEA	1	56.25%	70.2%	Present work
		HyperCel)
	5. Mixed-mode
		chromatogr. (PPA	1	52.50%	76.3%	Present work
		HyperCel)
	6. Mixed-mode
		chromatogr.	1	68.89%	84.0%	Present work
		(CaptoMMC)
	*Purity of the recovered rhApoA1 described based on densitometric analysis of the
	elution fraction on 12% polyacrylamide gel.

Table 3 .3. Peptides identified by ESI-MS/MS analysis of trypsinised rhApoA1

 3

	RT	Mass	Volume	Sequence	Seq Loc	Tgt mass	Δppm
	7.803 1501.6410	EFDEPPQSPWDR	A(1-12)	1501.6474 -4.27
	7.032 1426.6506	KWQEEMELYR	A(109-118)	1426.6551 -3.15
	7.580 1410.6537	KWQEEMELYR	A(109-118)	1410.6602 -4.60
	7.421 1298.5511	WQEEMELYR	A(110-118)	1298.5601 -6.99
	7.933 1282.5579	WQEEMELYR	A(110-118)	1282.5652 -5.72
	6.139 868.5083	QKVEPLR		A(119-125)	868.5131	-5.44
	6.830 1722.9331 QKVEPLRAELQEGAR	A(119-133)	1722.9377 -2.65
	6.439 612.3543	VEPLR		A(121-125)	612.3595	-8.45
	7.100 1466.7790 VEPLRAELQEGAR	A(121-133)	1466.7842 -3.52
	6.045 872.4314	AELQEGAR		A(126-133)	872.4352	-4.35
	5.873 1151.6273 QKLHELQEK	A(134-142)	1151.6299 -2.25
	6.008 1151.6228	QKLHELQEK	A(134-142)	1151.6299 -6.12
	6.021 895.4723	LHELQEK		A(136-142)	895.4763	-4.53
	6.157 895.4713	LHELQEK		A(136-142)	895.4763	-5.60
	7.025 1046.5024	LSPLGEEMR	A(143-151)	1046.5067 -4.06
	6.729 1317.6277	LSPLGEEMRDR	A(143-153)	1317.6347 -5.29
	5.940 1007.5575	ARAHVDALR	A(154-162)	1007.5625 -4.91
	6.140 780.4229	AHVDALR		A(156-162)	780.4242	-1.71
	7.059 1300.6379 THLAPYSDELR	A(163-173)	1300.6412 -2.50
	5.399 429.2688	LAAR		A(176-179)	429.2700	-2.67
	7.761 1814.8367 DSGRDYVSQFEGSALGK	A(26-42)	1814.8435 -3.77
	8.044 1399.6581 DYVSQFEGSALGK	A(30-42)	1399.6620 -2.73
	8.501 1611.7716 LLDNWDSVTSTFSK	A(48-61)	1611.7781 -4.02
	8.568 2201.1047 LREQLGPVTQEFWDNLEK	A(62-79)	2201.1117 -3.18
	8.940 2886.4404	LREQLGPVTQEFWDNLEKETEGL	A(62-85)	2886.4512 -3.74
			R
	8.813 1931.9205 EQLGPVTQEFWDNLEK	A(64-79)	1931.9265 -3.13
	9.166 2617.2561	EQLGPVTQEFWDNLEKETEGLR	A(64-85)	2617.2660 -3.80
	9.283 3220.5189	EQLGPVTQEFWDNLEKETEGLR	A(64-90)	3220.5347 -4.89
			QEMSK
	6.076 703.3473	ETEGLR		A(80-85)	703.3501	-3.90
	6.444 1306.6131	ETEGLRQEMSK	A(80-90)	1306.6187 -4.32
	7.436 2019.9696	ETEGLRQEMSKDLEEVK	A(80-96)	2019.9783 -4.28
	3.007 621.2742	QEMSK		A(86-90)	621.2792	-8.14
	6.711 1334.6305	QEMSKDLEEVK	A(86-96)	1334.6388 -6.18
	6.617 731.3692	DLEEVK		A(91-96)	731.3701	-1.33

Table 4 .1. Commonly employed strains of P. pastoris with phenotypes and

 4

	applications

Table 5 .1. Comparison of production parameters at various scales of expression

 5

	Parameter\Scale	Flask	2l Bioreactor	5l Bioreactor
	Volume of culture	150 ml	1800 ml	4000 ml
	Harvesting OD600	27.56	41.34	44.71
	Induction pH	6.0	5.0	5.0
	Buffering system	phosphate buffer	phosphate buffer + acid/base	phosphate buffer + acid/base
	Induction temperature	30°C	28°C	28°C
	rhApoA1 yield	22.4 mg/l	37.5 mg/l	43.8 mg/l

Table 5 .2. Comparison of purification efficiencies of packed column & EBA modes Scale Purification method Column size rhApoA1 content (mg) Yield (%) Purity of rhApoA1* Load Elution Packed column

 5

		Mixed-mode HEA HyperCel™	5ml prepacked column	2.24	1.26	56.25	70.2
	Packed column	Mixed-mode Capto MMC™	1ml prepacked column	2.21	1.51	68.89	84.0
	Scaled-up:	Mixed-mode Direct CST I	118ml settled-bed volume	113.48	84.77	74.69	79.0
	EBA (2 steps)	Anion exchange Resource Q	1ml prepacked column				96.2
	*Purity of the recovered rhApoA1 described based on densitometric analysis of SDS-PAGE.

  An in vivo evaluation of ApoA1Milano revealed that both the loss of Arginine as well as the addition of Cysteine contribute to its altered function(Alexander et al. 2009). Arginine-173 of wild type ApoA1 is potentially involved in an inter-helical salt bridge with Glutamate-169, which is disrupted in the Milano variant, thereby destabilising the helix bundle in turn modifying its lipid binding characteristics. However, the exact mechanism by which the Cysteine variants are of better quality isn't yet clear. Hence, in this thesis work, the two variants, ApoA1Milano and ApoA1Paris were generated to support future functional studies to better elucidate the mechanism of action of these variants. Agners, France). Competent E. coli DH5α cells (NEB5α) were purchased from New England Biolabs (Évry, France). All other chemicals for buffers and reagents were from Sigma-Aldrich (Saint-Quentin Fallavier, France).

	. The
	introduction of Cysteine in these two variants is hypothesised to increase
	stability of the resultant HDL molecule owing to homodimerization of ApoA1
	(Klon et al. 2000).
	6.1.2. STRUCTURAL CHANGES DUE TO POINT MUTATION AND POTENTIAL
	IMPACT ON FUNCTION
	A few studies have investigated the effects of a single point Arginine to Cysteine
	mutation in ApoA1. 6.2. EXPERIMENTAL
	6.2.1. MATERIALS
	Custom oligonucleotides for introducing site directed mutagenesis were
	synthesised from Eurogentec (

Chapter 2

Generation of wild-type rhApoA1 in P. pastoris

After verification by DNA sequencing, the construct was linearised by digestion with the restriction enzyme PmeI, and subsequently electroporated into competent P. pastoris X-33 cells as described in the methods section.

Subsequently, transformants were sequentially patched on to YPD agar plates containing increasing concentrations of Zeocin up to 2 mg/ml (Fig. 2.3). In addition to tolerance to 2 mg/ml Zeocin, all positive clones were also checked for the integration of the complete ApoA1 gene into the host genome. Colony PCR was performed on all clones resistant to 2 mg/ml Zeocin using ApoA1-specific primers under the conditions as detailed in the experimental section.

Subsequently, all reaction mixtures were subjected to 0.8% agarose gel electrophoresis (Fig. 2.4), which clearly confirmed the presence of ApoA1 gene in the transformed P. pastoris genome.

A B

Chapter 3

Purification of wild-type rhApoA1 expressed in P. pastoris by mixedmode chromatography

3.

After testing various concentrations of Triton X-114 and NaCl (Fig 3.1(a)), optimum recovery of rhApoA1 was achieved at 15% NaCl and 5% Triton X-114 (Fig. 3.1(b) panel iv, indicated by arrow). The combination of NaCl as electrolyte along with the non-ionic surfactant Triton X-114 has been shown to be effective in partitioning hydrophobic proteins from several sources [START_REF] Lopes | Cloud Point Extraction Applied to Casein Proteins of Cow Milk and Their Identification by Mass Spectrometry[END_REF]. In this case, the addition of NaCl helped achieve partitioning even at relatively lower concentrations (5% v/v) of Triton X-114.

After optimising conditions, the extraction method was scaled up to process 50 ml of P. pastoris broth containing rhApoA1. As evident in Despite the consistent reproducibility, eliminating the detergent from the surfactant rich phase was very difficult: dialysis, detergent exchange with CHAPS, on-column washing were attempted, but were unsuccessful. Washing with cold acetone was the only solution that eliminated the detergent (not completely) enough to analyse over electrophoresis. Hence, this process was

A B

Precipitation with cold acetone was highly effective in concentrating rhApoA1, which proved to be a useful precursor step to final polishing using anion exchange chromatography. Subsequent to the ion-exchange step, rhApoA1 was recovered with greater than 85% purity, with a few other additional higher molecular weight bands being present. Though this process was reproducible, utilisation of high concentrations of cold acetone (~ 60% acetone in the third precipitation step) coupled with extremely lengthy incubation periods (3 x 5 hrs) at sub-zero temperatures (-10°C) made this process unfeasible for scaling-up.

PURIFICATION OF rhApoA1 BY ION-EXCHANGE CHROMATOGRAPHY

As an improvisation to the method detailed above (Section 3.3.1.2), ion-exchange chromatography was applied directly to desalted P. pastoris expression broth containing rhApoA1. The purpose of this improvisation was to evaluate if the cold acetone treatment could be avoided, thereby reducing the processing time. literature. The comparative data clearly highlight the benefits and efficiencies of the mixed-mode chromatography based novel purification methods in capturing rhApoA1 from the P. pastoris expression broth, and their industrial applicability.

Among the three resins supported, Capto MMC showed most promise and ease of application in the single-step recovery of rhApoA1, with zero pretreatment of Pichia pastoris expression broth. Subsequently, this purification method has been scaled-up (detailed in Chapter 4), and enabled the development of an integrated process.

Mass spectrometric identification of the purified rhApoA1 molecule revealed a possible truncation in the C-terminal region of the protein. It was hypothesised that the truncation (if existent) would have been due to proteolytic activity of proteases secreted by P. pastoris into the medium. In order to verify this hypothesis, an additional comparative study was carried out, by expressing the rhApoA1 protein in a protease-deficient strain of P. pastoris: SMD 1168. This comparative study has been further discussed in Chapter 4 of this thesis.

Chapter 4

Cloning, Expression & Purification of rhApoA1 in P. pastoris protease deficient strain SMD-1168 4.

SDS-PAGE (Fig. 4.3) and stained with coomassie blue, which unequivocally demonstrated the successful expression of rhApoA1 in SMD-1168. Furthermore, rhApoA1 expressed and purified from X-33 was run as a reference protein to check for any apparent differences in molecular weight. A priori, there didn't seem to be a significant electrophoretic difference in molecular weight between the rhApoA1 expressed in X-33 and SMD-1168. Nevertheless, in order to unequivocally confirm the difference in size of the expressed rhApoA1 molecule, they were also analysed by mass spectrometry (Section 4.3.4).

PURIFICATION OF rhApoA1 BY MIXED-MODE CHROMATOGRAPHY

Upon successful expression of rhApoA1, ~15 ml of the expression broth was subjected to mixed-mode chromatography using CaptoMMC, as described in the experimental section (Section 4.2.4). Scale-up of production and purification of rhApoA1 using P. pastoris X-33

5.

number of processing steps and in establishing a continuous process [START_REF] Hjorth | Expanded-Bed Adsorption in Industrial Bioprocessing: Recent Developments[END_REF].

EXPANDED-BED ADSORPTION FOR THE PURIFICATION OF PROTEINS

Expanded bed adsorption (EBA) allows clarification and capture of target protein in a single step. In an expanded bed, a particulate adsorbent in a column is allowed to rise from its settled state by applying an upward flow, and the sample is injected from below [START_REF] Noubhani | On-Line Purification of His-Tag Enhanced Green Fluorescent Protein Taken Directly from a Bioreactor by Continuous Ultrasonic Homogenization Coupled with Immobilized Metal Affinity Expanded Bed Adsorption[END_REF]. Provided that the physical properties of the beads are significantly different from those of the particulates in the feedstock, the particulates can pass freely through the voids in the bed without becoming trapped [START_REF] Mattiasson | 10 Physicochemical Basis of Expanded-Bed Adsorption for Protein Purification[END_REF]. This eliminates the need for pre-clarification of crude feedstock before application to packed beds using centrifugal or filtration techniques and also permits the recovery of particulate bioproducts using column purification techniques. Subsequently, after all binding and wash is complete, the bed is allowed to settle, and the position of valve V1 is changed to downward flow, valve V2 is opened and Valve V3 is closed, permitting the eluted rhApoA1 protein to be collected.

This process could be further scaled to higher industrial levels, maintaining the same parameters. Paris-F: 5' AGATGCGCGACTGCGCGCGCGCCCA 3' Paris-R: 5' TGGGCGCGCGCGCAGTCGCGCATCT 3'

The mutagenesis was carried out using QuickChange ® II Site-Directed Mutagenesis Kit (Agilent Technologies), as per the manufacturer's instructions.

Briefly, a thermal cycling reaction was setup with template DNA (plasmid pPICZα-ApoA1), primers to incorporate mutations (Milano/Paris, as case may be), 10mM dNTPs and high-fidelity Pfu DNA polymerase and then subjected to thermal cycling: initial denaturation at 95ºC for 30secs, followed by 18 cycles of denaturation (95ºC, 30secs), annealing (55ºC, 1min), extension (72ºC, 5mins).

Subsequent to the thermal cycling, the template strand was digested by DpnI treatment, and the amplified fragments were transformed into competentNEB5α cells and plated on LB-agar plates containing 50µg/ml Zeocin. Plasmids were then isolated from transformed E. coli cells and verified for mutations by DNA sequencing (Millegen, Labège, France).

TRANSFORMATION AND SCREENING OF rhApoA1-Milano & rhApoA1-

Paris

After verification by DNA-sequencing, the constructs pPICZα-ApoA1-Milano and pPICZα-ApoA1-Paris were linearised by digestion with PmeI, and electroporated independently into competent P. pastoris X-33 cells as described earlier (Section 2.2.3). Subsequently, transforments were screened for their resistance to Zeocin up to 2mg/ml and verified for integration by colony PCR analysis as described earlier (Section 2.2.4).

FLASK-CULTURE EXPRESSION OF rhApoA1-Milano & rh-ApoA1-Paris

Three clones each of rhApoA1-Milano and rhApoA1-Paris were screened for their expression of the respective rhApoA1 mutant proteins. Single colonies of each clone were inoculated independently in 10ml BMGY containing 200μg/ml Zeocin as a preculture. After overnight growth at 30°C and 200rpm, the precultures were inoculated into 140ml of fresh BMGY (buffered glycerol complex media). After about 24hrs, when the cells had grown to a reasonable quantity (based on their OD at 600nm), they were centrifuged and resuspended in 150ml of BMMY (buffered methanol complex media). Induction was carried 6.3. RESULTS

GENERATION OF rhApoA1-Milano & rhApoA1-Paris

The construct pPICZα-ApoA1 was subjected to site-directed mutagenesis as described in the experimental section, and the resulting clones (pPICZα-ApoA1-Milano and pPICZα-ApoA1-Paris) were verified by DNA sequencing. Upon transformation into competent P. pastoris X-33 cells, transformants were screened for their resistance to Zeocin up to 2mg/ml (Fig. 6.2). Clones that showed consistent resistance to 2mg/ml Zeocin were further verified for integration of the mutated ApoA1 gene by colony PCR analysis (Fig. 6.3). Three colonies each from Milano and Paris transformants were further screened for preliminary expression studies in shake flasks.

FLASK-CULTURE EXPRESSION OF rhApoA1-Milano & rh-ApoA1-Paris

Three colonies transformed with ApoA1-Milano gene (A7, B6, E4) and three colonies transformed with ApoA1-Paris gene (A2, B3, E1) were subjected to preliminary expression studies in shake flasks, along with a vector transformed P. pastoris clone as a negative control (ZαA) as described in the methods section.

The OD600nm of cells were periodically monitored for growth, and samples drawn at various time points were analysed by dot-blot analysis using anti-ApoA1 polyclonal antibodies. All six clones exhibited consistent growth as evidenced by the absorbance at 600nm (Fig. 6.4). Production of rhApoA1 variants was confirmed by dot-blot analysis (Fig. 6.5), with an absence of signal prior to induction and with a constant rise in intensity of signal with increasing duration of induction. There was no signal corresponding to the vector-transformed X-33 cells (Fig 6 .5 -lane C), verifying the specificity of the polyclonal antibodies used for the detection of ApoA1 and its variants. Purified wild type rhApoA1 (Fig. 6.5 -position '+')

showed a clear signal, further validating the obtained results.

The chromatographic profile obtained with rhApoA1-Milano (Fig. 6.8(a)) was similar to that of wild type rhApoA1. Nevertheless, the protein (rhApoA1-Milano) was more strongly bound and was eluted only at pH 3.0 (Fig. 6.8(b), lane A3). This stronger binding does indicate towards an altered conformation of the folded rhApoA1-Milano protein with increased hydrophobicity, vis-à-vis its wild type counterpart. Although the target protein was recovered in part by elution at pH 3.0, there were several other protein bands present in the lane (Fig. 6.8(b), lane A3) which required further polishing prior to recovering the protein.

Secondly, the mixed mode resin CaptoMMC was evaluated for the recovery of rhAPoA1-Milano. Binding for all CaptoMMC experiments were at pH 4.5. It is well documented that both pH and conductive strength of the microenvironment strongly dictate binding to multi-modal ligands (Pezzini et al. 2015). Hence, in this study, two strategies were tested for the recovery of rhApoA1-Milano from P. pastoris expression broth: (i) by raising the pH while maintaining conductivity, followed by a rise in conductivity; and (ii) by eliminating the conductivity, followed by a rise in pH. By the first approach, the bound rhApoA1-Milano was efficiently recovered by raising the pH without eliminating the salt (Fig. 6.9, peak #1). However, a number of additional bands were present, which required further polishing in order to obtain our target protein in a pure form. A second concern was the Both variants (rhApoA1-Milano and rhApoA1-Paris) require further polishing to recover them in homogeneity for future functional analyses. The bioprocess for the production of these two variants could pave way for a more efficient method for producing them for eventual therapeutic applications.

Chapter 7

General Conclusions and Perspectives

This thesis has worked towards the development of a method for efficiently producing wild type and variants of human apolipoprotein A-I in P. pastoris.

Production of recombinant ApoA1 has been previously described using different systems. The bacterial system E coli presented the disadvantage of instability of mRNA and rapid degradation of mature ApoA1 [START_REF] Isacchi | Mature Apolipoprotein AI and Its Precursor proApoAI: Influence of the Sequence at the 5' End of the Gene on the Efficiency of Expression in Escherichia Coli[END_REF]. Another disadvantage was the use of tags to facilitate purification steps [START_REF] Schmidt | Expression and Purification of Recombinant Human Apolipoprotein A-I in Chinese Hamster Ovary Cells[END_REF][START_REF] Bergeron | Characterization of Human Apolipoprotein A-I Expressed in Escherichia Coli[END_REF]): despite the presence of these tags, the purification steps require many treatment such as cell lysis, protein precipitation with ammonium sulphate, delipidation, endotoxin clearance at the end of the purification, in addition to an enzymatic cleavage of the affinity tag. In some cases the protein is mainly found into inclusion bodies, which further complicates the purification strategy [START_REF] Angarita | Role of Urea on Recombinant Apo A-I Stability and Its Utilization in Anion Exchange Chromatography[END_REF].

Another reported approach was the production of Apolipoprotein A-I using baculovirus-insect cell expression system [START_REF] Moguilevsky | Correct in Vivo Processing of a Chimeric Ubiquitin-Proapolipoprotein A-I Fusion Protein in Baculovirus-Infected Insect Cells[END_REF]. In this case, it was necessary to express it in the proapolipoprotein form in order to ensure the secretion of the protein. Without the proapoA-I sequence, most of the mature apolipoprotein A-I was found in the cytoplasm and only a small proportion of ApoA-I was secreted into the culture medium.

The production of Apolipoprotein A-I has also been evaluated in Chinese

Hamster Ovary Cells [START_REF] Schmidt | Expression and Purification of Recombinant Human Apolipoprotein A-I in Chinese Hamster Ovary Cells[END_REF]). However, typical yields were only 0.5 to 1 µg/mL Apolipoprotein A-I was secreted by CHO in a serum-free culture medium, and upto 10 µg/mL after several optimisation. Higher concentrations were also achieved, however, immunoblot analysis revealed a carboxy-Terminal proteolysis.

Few studies have used the Pichia pastoris expression system to produce apolipoprotein A-I [START_REF] Feng | High Yield and Secretion of Recombinant Human Apolipoprotein AI in Pichia Pastoris[END_REF]). In the present study, the focus was on the production of apolipoprotein A-I exploiting P. pastoris' ability to produce and secrete recombinant proteins with high yield. A high expressing clone was selected after screening with zeocin, and production yields of 20 mg/L was