Dragana P C ; De Barros
email: dragana@ist.utl.pt

Joaquim M. S.; Cabral

Luís P Fonseca

Joaquim M S Cabral

Clemens K Weiss

Katharina Landfester

Synthesis of alkyl esters by cutinase in miniemulsion and organic solvent media

Keywords: alkyl esters, miniemulsion, organic solvent, cutinase alkyl esters, miniemulsion, organic solvent, cutinase

The main objective of this work was to study and test the nature and influence of reaction media (organic solvent vs. miniemulsion system) on the synthesis of alkyl esters catalyzed by Fusarium solani pisi cutinase. Ester synthesis and cutinase selectivity for different chain length of acids and alcohols (ethyl and hexyl) were evaluated.

In iso-octane cutinase exhibits rates of esterification after one hour between 0.24 µmolmg -1 min -1 for ethyl oleate and 1.15 µmolmg -1 min -1 for ethyl butyrate, while in miniemulsion system were from 0.05 µmolmg -1 min -1 for ethyl heptanoate to 0.76 µmolmg -1 min -1 for ethyl decanoate. The reaction rate for the synthesis of hexyl esters in miniemulsion system was from 0.19 µmolmg -1 min -1 for hexyl heptanoate to 1.07 µmolmg -1 min -1 for hexyl decanoate.

High conversion yields of 95 % at equilibrium after 8h of reaction in iso-octane for pentanoic acid (C 5 ) with ethanol at equimolar concentration (0.1M) was achieved.

Additionally, this work also shows that a significant and unexpected change in cutinase selectivity occurred for longer chain length carboxylic acids (C 8 -C 10 ) in miniemulsion system comparing with organic solvent (iso-octane) and previous studies in reverse micellar systems where a selectivity towards short chain fatty acids ( C 4 -C 6 ) was observed. The possibility to work with higher concentration of substrates ,without inhibitiory effect on enzyme, was another advantage of miniemulsion system.

Introduction

Alkyl esters belong to the group of flavor compounds that are very important parts of aromas, applied in food, beverage, cosmetic and pharmaceutical industries. The esters of short chain acids (C 6 -C 10 ) and alcohols (C 2 -C 6 ) are well-known flavor and fragrance compounds. These compounds have odors of different fruits and are parts of complex mixtures providing specific aromas. Longer chain acids esters (C 18 ) are used in cosmetic industry as emollient for personal care applications [START_REF] Hills | Industrial use of lipase to produce fatty acid esters[END_REF].

Enzymatic synthesis of flavor esters is an interesting and innovative process for aroma production for current and future industrial applications.

Until now the aroma esters were generally produced via the chemical catalytic or by extraction from natural sources. The products chemically synthesized are characterized by low purity and they are not classified as natural products. On the other hand natural aromas obtained by the extraction are very expensive and their quality very often depends on climate and geographic conditions. Specificity and selectivity of biocatalysts are providing the possibility of working at mild reaction conditions, without undesirable side reactions, generating the product with high degree of purity. As a further benefit the products are no racemic mixtures but usually only one stereoisomer. These are the main advantages of using enzymes in new industrial flavor manufacture processes [START_REF] Hari-Krishna | Lipases and lipase-catalyzed esterification in non-aqueous media[END_REF][START_REF] Berger | Aroma Biotechnology[END_REF][START_REF] Turner | Recent Advances in the use of enzyme-catalysed reactions in organic synthesis[END_REF].

Moreover, the increasing sensitivity today for environmentally friendly process F o r P e e r R e v i e w 4 and preference for "green" technology and "natural" products favor the synthesis of flavor compounds via biotechnology route [START_REF] Serra | Biocatalytic preparation of natural flavours and fragrances[END_REF][START_REF] Schrader | Applied biocatalysis for the synthesis of natural flavour compounds-current industrial processes and future prospects[END_REF].

Lipases are mostly used as biocatalyst for the reactions of hydrolysis, esterification and transesterification. Lipases are characterized by their catalytic action preferably at the interface between water and an insoluble substrates or organic solvent containing the reactatns. Another interesting group of hydrolytic enzymes are cutinases, also which belong to the class of serine esterase as most of the lipases do [START_REF] Gandhi | Lipase-catalyzed esterification[END_REF][START_REF] Cabral | Biocatalysis in Organic media by cutinase in Reversed micelles and zeolites[END_REF] but in comparison to the lipase they are not characterized by the phenomenon of interfacial activation.

Industrially, lipases were first used in hydrolytic processes in aqueous media, but increasing interest focuses on their potential use as biocatalysts for new processes in various reaction media due to their great importance and role for esterification reactions. Thus, the esterification reactions were performed in non-conventional media such as organic solvents (biphasic systems, reverse micellar, and monophasic system) [START_REF] Gandhi | Lipase-catalyzed esterification[END_REF], supercritical fluids [START_REF] Vija | Lipaze-catalyzed esterification in supercritical carbon dioxide and in hexane[END_REF], ionic liquids [START_REF] Lozano | Enzymatic esters synthesis in ionic liquids[END_REF], aqueous twophase systems [START_REF] Janssen | Enzymatic synthesis of. carbohydrate esters in aqueous media[END_REF], or solid-gaseous media [12]. There are several advantages in using organic solvent as reaction media such as increased solubility of nonpolar substrates and products, shifting thermodynamic equilibrium to favor the ester synthesis in relation to hydrolysis, and the minimization of enzyme inhibition by substrates or products, etc. Still, there are disadvantages of using organic media, which can lead to denaturation or inhibition of the enzyme, separation of solvent from the product, lack of green solvent, and their compatibility with the demands and regulations of FDA and other official regulatory agencies [START_REF] Serra | Biocatalytic preparation of natural flavours and fragrances[END_REF]. Supercritical properties [START_REF] Vermuë | Biocatalysis in non-conventional media: medium engineering aspects[END_REF][START_REF] Cernia | The role of the reaction medium in lipasecatalyzed esterification and transesterification[END_REF]. Recently, ionic liquids have been explored as alternative for organic solvent, but this investigation still has to be approved in future [START_REF] Cantone | Biocatalysis in non-conventional media-ionic liquids, supercritical fluids and gas phase[END_REF][START_REF] Moniruzzaman | Water-in-ionic liquid microemulsions as a new medium for enzymatic reactions[END_REF].

As in esterification reactions two products, ester and water are generated, the influence and accumulation of water in the reaction medium has significant influence on the chemical equilibrium. For successful high-yield esterification the water has to be removed from the site of the reaction. Different techniques were used for water removal to shift the equilibrium from reactants to products [START_REF] Gubicza | On-line water removal during enzymatic asymmetric esterification in organic media[END_REF][START_REF] Deng | Enzymatic production of fatty acid alkyl esters with a lipase preparation from Candida sp. 99-125[END_REF].

Irrespective of the limiting aspect of water in esterification reactions, successful efforts have been put in using water as a (green) reaction medium for enzyme transformations [START_REF] Ran | Recent application of biocatalysis in developing green chemistry for chemical synthesis at the industrial scale[END_REF][START_REF] Wei | Water as reaction medium for clean chemical processes[END_REF].

Miniemulsions are heterophase systems consisting of uniform, small droplets in a continuous phase characterized by high stability. They are obtained using intensive shearing force. A typical miniemulsion system used for an esterification reaction is composed of an oil phase (as substrates) containing a hydrophobic agent (e.g. hexadecane), an emulsifier (usually non-ionic surfactant) stabilizing the oil reactant droplets, and water. The mixture is homogenized by high shear (e.g. ultrasonification) to obtain homogenous and monodisperse droplets in the size range of about 200 nm dispersed in water [START_REF] Landfester | Synthesis of colloidal particles in miniemulsions[END_REF][START_REF] Landfester | Polyreactions in miniemulsions[END_REF]. Esterification is performed by the addition of an enzyme as a biocatalyst dissolved in the aqueous phase Fig 1 .   Lipase catalyzed esterification is well established [START_REF] Aschenbrenner | Enzymatic esterification in aqueous miniemulsion[END_REF] and was shown to be highly efficient in the production of long chain phenylalkanoates at mild reaction conditions.. The objectives of the presented studies are to investigate and compare the conditions of the enzymatic synthesis of flavor esters by Fusarium solani pisi cutinase produced by Sacharomyces cerevisiae SU50 in two different media: first, organic media, in particular iso-octane, an organic solvent recognized as safe ingredient in food and beverage industrial processes by FDA, and second in miniemulsion system allowing to use water as reaction medium for ester synthesis.

MATERIALS AND METHODS

Materials

Cutinase from Fusarium solani pisi was produced as described previously [START_REF] Calado | Development of a Fed-Batch Cultivation strategy for the Enhanced Production and secretion of Cutinase by a Recombinant Saccharomyces cerevisiae SU50 Strain[END_REF][START_REF] Calado | Effect of Saccharomyces cerevisiae fermentation conditions on expanded bed adsorption of heterologous cutinase[END_REF] and used for enzymatic esterification. Reagents including acetic (96%, ACROS, Belgium), butyric (99.5%, FLUKA, Germany), pentanoic (99.5%, FLUKA, Germany) , hexanoic (98%, FLUKA, Germany), heptanoic, octanoic (99%, SIGMA, Germany), decanoic and oleic acid (FLUKA, Germany), and ethanol abs.

(Merck, Germany) and hexanol (Merck, Germany) were used for ester synthesis while hexadecane 99% (SIGMA, Germany) and LutensolAT-50 powder (donation from BASF, Germany) were used to obtain miniemulsion system. All other chemicals used were analytical grade. NaOH (Merck, Germany) as reagent and tetrahydrofuran (THF) (Merck, Germany) as solvent for titration were used. Iso-octane (Sigma-Aldrich, Germany) was used in the reaction medium and ndecane (VWR, Germany) was used as internal standard for Gas Chromatography (GC) analysis. Anhydrous sodium sulfate, in powder form (Sigma-Aldrich,

Germany), was used to dry samples from organic solvent after esterification and before GC analysis.

Production of cutinase

The F. solani pisi cutinase wild-type was produced by recombinant S. cerevisiae SU50 strain. The cutinase producing S. cerevisiae SU50 strain (Mata, leu2-3, ura3, gall:URA3, ML-S, MAL3, SUC3) contains the expression vector pUR7320 constructed and provided by the Unilever Research Laboratory, Vlaardingen, the Netherlands. The strain was stored at -80°C in frozen tubes containing selective medium and 50% (v/v) glycerol (Merck, Germany) [START_REF] Calado | Development of a Fed-Batch Cultivation strategy for the Enhanced Production and secretion of Cutinase by a Recombinant Saccharomyces cerevisiae SU50 Strain[END_REF].

Purification of cutinase and production of lyophilized enzyme preparation

The isolation and purification of cutinase excreted by recombinant S. cerevisiae SU50 strain was carried out by expanded bed adsorption (EBA) [START_REF] Calado | Effect of Saccharomyces cerevisiae fermentation conditions on expanded bed adsorption of heterologous cutinase[END_REF]. Frontal adsorption experiments were carried out in an EBA column Streamline 25 with a settled bed adsorbent height of 15 cm and the column top adjusted to 45 cm. The cation adsorbent Streamline SP XL from Amersham Pharmacia Biotech, Sweden was used to isolate the cutinase directly from the fermentation broth. The cutinase activity and protein concentration of the collected effluent fractions from the column were determined after each run. The pool of elution fractions exhibiting highest cutinase activity were previously dialyzed against 20mM phosphate buffer pH 7.0 and then frozen at -80°C and lyophilized (B. Braun Biotech. International Specific activity of lyophilized cutinase preparations was determined as 170 U/mg and samples of these enzyme preparations when analyzed with gel electrophoresis showed only one band of 22kDa, i.e., pure cutinase [START_REF] Sambrook | Molecular cloning, a laboratory manual[END_REF].

Enzymatic esterification reaction in miniemulsion system

Unless otherwise stated a typical esterification reaction was carried out in 30 ml capped flasks used as reactor with 20 ml of working volume with equimolar substrate concentration (Table 1). Alcohol, acid, hexadecane, water and Lutensol, (relative amounts see Table 2) are homogenized by magnetic stirring for 1 h. The emulsion was ultrasonicated for 120 s with pulses of 5 s and pauses of 10 s at 70 % amplitude (Branson sonifier W450 digital, tip size 6.5 mm) with ice cooling. 20 ml of the miniemulsion was added into the reaction vessel containing the appropriate amount of enzyme (5 mg/ml of reaction mixture). The reaction was performed in thermostated incubator (Advanced ChemTec PLS 4x4, 400 rpm) at 40 °C unless otherwise stated. This setup enabled parallel experiments by simultaneous use of 5 reactors against at least one blank without enzyme.

Samples were withdrawn periodically (20µl) using a needle (as to preserve the rubber cap) and then mixed with NMR solvent (DMSO-d 6 ) (5ml) prior to NMR analysis.

Determination of esterification degree in miniemulsion system

Analysis of reaction mixture was determined by liquid 1H NMR spectroscopy (Bruker 400 MHz) using DMSO-d 6 (Deutero GmbH, Germany) as solvent. The esterification yield has been calculated from the ratio of the area of the methylene protons adjacent to the carboxyl-group ( α ´CH 2 ) at 2.26 ppm to the sum of the areas of ester ( α ´CH2) at 2.26 ppm and acid protons ( 2 CH 2 ) at 2.18 ppm defined as in Eq. ( 1). The sum of ester and acid concentrations was equal to the initial acid concentration.

Eq. (1):

An example for 1 H NMR spectra for synthesis of ethyl decanoate is shown in Fig.

2.

Titration of free acids in reaction media (Schott Titronic, Germany) was used as control method for monitoring the progress of esterification. At given incubation periods, aliquots of 100µl of the reaction mixture were withdrawn using a syringe and were immediately diluted in 300µl THF in order to stop the enzyme action.

The degree of esterification was monitored by determination of residual acid content by titration against sodium hydroxide (0.1M NaOH) using phenolphthalein as an indicator.

Esterification catalyzed by lyophilized cutinase in iso-octane

The esterification of acid and alcohol at equimolar concentration (0.1M) catalyzed by cutinase was also carried out in iso-octane, as reaction medium because it was observed that the enzyme is inhibited by substrate concentrations higher than % esterification = Samples were withdrawn periodically using a needle (as to preserve the rubber cap) and then dried prior to GC analysis with anhydrous sodium sulfate. The conversion yield was calculated according to the molar ratio between ethyl ester and respective acid.

Monitoring and determination of esterification degree in iso-octane

The concentration of ethanol, carboxylic acids (acetic acid, pentanoic acid, hexanoic acid, octanoic acid, decanoic acid, oleic acid) and ethyl esters (ethyl acetate, ethyl pentanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl Ethyl acetate: oven temperature was held at 50ºC for 4 min before being elevated to 150ºC for 3.67 min at 30ºC/min; injector temperature was set at 200 ºC; detector temperature was set at 250 ºC; carrier gas was nitrogen.

Ethyl butyrate: oven temperature was held at 50ºC for 4 min before being elevated to 150ºC for 2.34 min at 15ºC/min; injector temperature was set at 200 ºC; detector temperature was set at 250 ºC; carrier gas was nitrogen.

Ethyl pentanoate: oven temperature was held at 50ºC for 4 min before being elevated to 170ºC for 2 min at 30ºC/min; injector temperature was set at 200 ºC and detector temperature is set at 250 ºC; carrier gas was nitrogen.

Ethyl hexanoate: oven temperature was held at 50ºC for 4 min before being elevated to 160ºC for 1.67 min at 15ºC/min; injector temperature was set at 200 ºC; detector temperature was set at 250 ºC; carrier gas was nitrogen.

Ethyl octanoate: oven temperature was held at 50ºC for 4 min before being elevated to 180ºC for 3.67 min at 30ºC/min; injector temperature was set at 200 ºC; detector temperature was set at 250 ºC; carrier gas was nitrogen. Ethyl decanoate: oven temperature was held at 50ºC for 4 min before being elevated to 190ºC for 4.34 min at 30ºC/min; injector temperature was set at 200 ºC; detector temperature was set at 250 ºC; carrier gas was nitrogen.

Ethyl oleate: oven temperature was held at 50ºC for 4 min before being elevated to 190ºC for 5.34 min at 30ºC/min; injector temperature was set at 200 ºC; detector temperature was set at 250 ºC; carrier gas was nitrogen.

. Esterification in miniemulsion system

This studies on the synthesis of alkyl esters using cutinase were based on previous work carried out by Aschenbrenner et al. [START_REF] Aschenbrenner | Enzymatic esterification in aqueous miniemulsion[END_REF]. The authors successfully established a miniemulsion system for the synthesis of monoesters of linear carboxylic acids with ω-phenyl alkanoles using various lipases ( Chirazyme L-5 from Candida antartica, Lipase G from Penicilium cammemberti, Lipase PS from Pseudomonas cepacia). As this system has shown to yield high conversions in very short reactions times at mild conditions, the range of substrates was extended to linear alcohols. Various carboxylic acids including hexanoic (C 6 ), heptanoic (C 7 ), octanoic (C 8 ), decanoic (C 10 ) and oleic acid (C 18 ), and two short chain alcohols ethanol (C 2 ) and hexanol (C 6 ) were used for enzymatic esterification. allows to obtain a miniemulsion system. Nevertheless the system is rather unstable as few minutes after addition of the enzyme and start of the esterification reaction, phase separation occurred and the reaction stopped. The water solubility of the short chain acids is too high, so the acid will diffuse from the droplet into the aqueous phase. This observation is consistent with the results obtained by Aschenbrenner et al., who also observed phase separation in systems with acid substrates shorter than seven C-atoms. This is probably due to the fact that acids with lower chain lengths can act as cosurfactants at the oil/water interface and lower the interfacial tension so that the droplet size decreases or do not allow the formation of stable emulsion droplets, in spite of using a concentration of Lutensol of 1.62% (w/w), the surfactant in the present miniemulsion system (Table 2). Additionally, these short acids are also characterized by low viscosity and hydrophobicity that allows diffusion from the emulsion droplets into the water phase [START_REF] Manabe | Dehydration reaction in water. Brønsted acid-surfactant-combined catalyst for ester, ether, thioehter, and dithioacetal formation in water[END_REF].

With other acids with chain length lower than C 6 it was not possible to obtain

The acids with longer chain length than C 6 and the two alcohols tested in the miniemulsion systems have different solubility in water and, consequently, different capacity to be encapsulated into the emulsion droplets. Ethanol is completely miscible with water, whereas hexanol is only slightly soluble in water (590 mg/100g at 25°C) [START_REF]cooperation between the International Programme on Chemical Safety and the Commission of the European Communities © IPCS, CEC[END_REF] while the solubility of the acids decreases with increasing chain length (Table 3). According to these values of solubility it is possible to conclude that almost all of the acids and hexanol were located inside 3). Furthermore cutinase presented an extraordinary synthetic capacity in the miniemulsion system with optimized composition (Table 2) which was carried out previously by other enzyme [START_REF] Aschenbrenner | Enzymatic esterification in aqueous miniemulsion[END_REF].

The main goal of this work was thus to explore the esterification potential of cutinase for these types of substrates ( as named previously), and to assess the possibility to work with high substrates concentrations ( Table 1). The influence of different concentration of substrates on reaction rate and mass transport phenomena will be studied and published in following work.

The conversion rates of ester synthesis by cutinase in miniemulsion show a direct relationship to increasing acid chain length with a maximum for decanoic acid (C 10 ). This could be observed for both alcohols applied (ethyl esters and hexyl esters) with rates of 0.76 and 1.07 µmolmg -1 min -1 for ethanol and hexanol, respectively obtained in the first hour of reaction (Fig 3).

These results show an interesting and unexpected shift of cutinase specificity to higher chain length of acids (C 10 ) when compared with results obtained for reactions of esterification and transesterification performed in reverse micellar system. Here the short chain acids (C 4 -C 6 ) were esterified with higher specificity [START_REF] Pinto-Sousa | Ester synthesis by a recombinant cutinase in reversed micelles of a natural phospholipid[END_REF][START_REF] Sebastião | Synthesis of fatty acid esters by a recombinant cutinase in reversed micelles[END_REF][START_REF] Cunnah | Esterification and transesterification catalysed by cutinase in reverse micelles of CTAB for the synthesis of short chain esters[END_REF]. On other hand, cutinase showed higher esterification reaction specificity for hexanol than ethanol confirming the results obtained already in reverse micellar systems [START_REF] Pinto-Sousa | Ester synthesis by a recombinant cutinase in reversed micelles of a natural phospholipid[END_REF][START_REF] Sebastião | Synthesis of fatty acid esters by a recombinant cutinase in reversed micelles[END_REF][START_REF] Cunnah | Esterification and transesterification catalysed by cutinase in reverse micelles of CTAB for the synthesis of short chain esters[END_REF] The only exception was found for ethyl oleate and hexyl oleate where rate of ester synthesis was almost equal (0.33 and 0.29 µmolmg -1 Deleted: F o r P e e r R e v i e w 16 min -1 for ethyl and hexyl esters respectively). Thus, it is possible to conclude that miniemulsion system forms a different microenvironment for the enzyme that changes slightly the cutinase specificity in favor of the longer chain of acids (C 8 -C 10 ) and not for specific type of alcohol.

The esterification reactions in miniemulsion system were monitored during 24h

showing that the equilibrium was definitively achieved for all esters after 8h. The specific time, for which maximum conversion could be detected, depends on the chain length of acid and alcohol. For instance, for ethyl esters the equilibrium for ethyl decanoate and ethyl oleate was achieved after 2h, and with smaller chain length of ethyl esters, the maximum of conversion was at 1h. The reaction equilibrium for hexyl octanoate was reached after 2h, and for hexyl decanoate and hexyl oleate after 8h of reaction. However it is possible that low conversions for shorter chain ethyl esters can be attributed to the instability of the respective miniemulsion system. Phase separation and destabilization of the miniemulsion during the synthesis of hexyl heptanoate was observed leading to the low conversion yield termination of the reaction after 1h.

The profile of conversion yields at the equilibrium was very similar for ethyl and hexyl esters but the latter generally shows higher conversion yields in compared to ethyl esters (Fig. 4). The maximum conversion yields were found for decanoic acid (C 10 ) for both ethyl (59%) and hexyl (76%) esters. The conversion yields for ethyl heptanoate (C 7 ) and octanoate (C 8 ) were very low 1% and 5%, respectively and the synthesis of ethyl hexanoate (C 6 ) was not detectable. Conversely, for oleic acid (unsaturated C 18 ), the conversion at equilibrium was 40% (Fig. 4). Regarding hexyl esters, shorter chain acids also gave lower conversion yield not detectable for hexyl hexanoate, and of 10, 52 and 76% for heptanoate, octanoate and decanoate, respectively. Also the esterification of hexanol with oleic acid (C 18 ) by cutinase gives an unexpectedly high conversion yield at equilibrium of 69% (Fig. 4).

These results confirm the observations by Manabe et al. [START_REF] Manabe | Dehydration reaction in water. Brønsted acid-surfactant-combined catalyst for ester, ether, thioehter, and dithioacetal formation in water[END_REF] of chemical synthesis dehydrative esterification, that hydrophobic and lipophobic substrates favor esterification reactions in miniemulsion systems, probably due to the formation of very stable hydrophobic droplets. Additionally, due to their nanosize that allow a huge reaction surface and the water, which is formed by esterification inside of the droplets, is directly expelled out of the hydrophobic droplets even in presence of a large amount of water used as continuous phase [START_REF] Manabe | Dehydration reaction in water. Brønsted acid-surfactant-combined catalyst for ester, ether, thioehter, and dithioacetal formation in water[END_REF]. Thus, compared to completely water soluble reactants as acids of short chain length, the equilibrium of the reaction with longer and high lypophilic acids is displaced in favor of ester formation.

Cutinase used in a similar type of miniemulsion system shows potential not only for hydrophobic substrates (carboxylic acids, C 10 and C 18 ) but also for enzymatic synthesis of hydrophilic (ethanol) substrates, and partly hydrophilic substrates (hexanol, carboxylic acids from C 6 to C 8 ). In more detailed studies it will be necessary to optimize the miniemulsion composition especially the amount of hydrophobic agent and surfactant.

The low conversion yield and the low initial conversion rate for ethyl esters may be the result of the high solubility of ethanol in the continuous phase water and a F o r P e e r R e v i e w 18 strong inhibition effect of ethanol on cutinase activity. This is due to the fact that ethanol is more polar than hexanol, and its concentration in the aqueous phase is high. This could induce stripping of essential water from the enzyme environment, resulting in an inhibition effect.

Esterification in iso-octane

Iso-octane as an adequate esterification reaction medium for ethyl ester formation from different carboxylic acids with different chain lengths as acetic (C 2 ), butyric (C 4 ), pentanoic (C 5 ), hexanoic (C 6 ), octanoic (C 8 ), decanoic (C 10 ) and oleic acid (unsaturated, C 18 ) was also investigated. Reactions were performed with equimolar concentration of the substrates (0.1M) at 30ºC. The higher concentration of substrates showed inhibition effect on cutinase activity in the esterification in iso-octane (data not shown).

A maximal rate of ethyl ester formation (V, µmolmg -1 min -1 ) catalyzed by cutinase was obtained for butyric acid (C 4 ) and the minimal values was found for acetic (C 2 ) and oleic (C 18 ) acid, with the extreme of chain lengths tested in this work.

The results are in partial agreement with those reported by Sebastião et al. in reversed micellar system [START_REF] Calado | Development of a Fed-Batch Cultivation strategy for the Enhanced Production and secretion of Cutinase by a Recombinant Saccharomyces cerevisiae SU50 Strain[END_REF] (Fig. 5). The exception is that in reversed micellar systems, cutinase does not present a significant activity on oleic acid. Although, esterification reactions performed by cutinase in iso-octane presents a "lag "phase which is the time that cutinase needs before starting to be detected and quantified, the formation and accumulation of ester in the reaction media (Figure 5) and this The esterification reactions for different chain length of acids at these experimental conditions reaches their equilibria after 8h with a maximum value for ethyl pentanoate (95%) and high yields for almost all the other ethyl esters, such as acetate (88%), butyrate (86%), hexanoate (64%), octanoate (91%), decanoate (82%) and lowest value for oleate (43%). The rates of esterification after 1h for ethyl hexanoate and ethyl octanoate were not significantly different (Fig. 5), but the final conversion yield for ethyl hexanoate was slightly lower maybe due to some inhibitory or deactivation effect of hexanoic acid on cutinase activity (Fig. 6). Hexanoic acid inhibitory effect at concentration of 0.1M and cutinase specificity for short chain acids was observed by Cunnah et al. [START_REF] Cunnah | Esterification and transesterification catalysed by cutinase in reverse micelles of CTAB for the synthesis of short chain esters[END_REF] in CTAB reverse micellar system. Significant drop in conversion yield (Figure 6) between butyric acid and hexanoic acid may be results of cutinase specificity, was shown by previous work too [START_REF] Cunnah | Esterification and transesterification catalysed by cutinase in reverse micelles of CTAB for the synthesis of short chain esters[END_REF] . At this substrate concentration octanoic acid shown less inhibitory effect on cutinase. Still, these results confirm the selectivity of cutinase for shorter chain acids (C 4 -C 6 ) and iso-octane shows to be an adequate organic solvent to perform enzymatic esterifications.

Concluding remarks:

The enzymatic esterification of ethyl-and hexyl esters by cutinase in two different reaction media, a miniemulsion system and iso-octane, was demonstrated. Cutinase shows a distinct selectivity for short chain ethyl esters in iso-octane confirming the results obtain by previous authors [START_REF] Pinto-Sousa | Ester synthesis by a recombinant cutinase in reversed micelles of a natural phospholipid[END_REF][START_REF] Sebastião | Synthesis of fatty acid esters by a recombinant cutinase in reversed micelles[END_REF][START_REF] Cunnah | Esterification and transesterification catalysed by cutinase in reverse micelles of CTAB for the synthesis of short chain esters[END_REF] as the maximum of cutinase activity was exhibited for ethyl butyrate (C 4 ) and pentanoate (C 5 ).

Enzymatic synthesis in an organic solvent (iso-octane) still favors the synthesis of short chain alkyl esters and good esterification yields were obtained (ethyl butanoate 86%, ethyl pentanoate 95%).

Miniemulsion systems also showed to have a great potential as a reaction media for synthesis of ethyl-and hexyl esters catalyzed by cutinase. The cutinase was more tolerant for high substrate concentration , comparing with the iso-octane as reaction medium. Interestingly the syntheses are characterized by a shift of the enzyme selectivity towards carboxylic acids with longer chain length especially decanoic acid (C 10 ) and additionally surprisingly good activity on oleic acid (C 18 ).

It was interesting to verify and prove with this work that cutinase catalyzed esterification reaction occurred in a miniemulsion system and surprisingly led to excellent conversion yields of 59 and 76% after 8h for decanoic acid (C 10 ) with ethanol and hexanol, respectively, applied with the 4 to 10 fold concentration substrates than in iso-octane. This is only possible because of the accumulation of substrates in miniemulsion droplets prevents direct contact with the enzyme as when used in iso-octane as reaction media. This prevents the equilibrium to be shifted back to the educts by hydrolysis of the ester.

These results underline that the miniemulsion system has perspective for the synthesis of longer chain alkyl esters (C 18 ), and as the reaction occur mainly in water ("clean solvent"), it is a very interesting approach for the application for

  used in reaction of enzymatic synthesis regarding their physical

8 CHRIST

 8  overnight. Lyophilized pure cutinase was stored deep-frozen at -20°C before used in esterification reactions.2.4 Characterization of the cutinase preparationsThe cutinase estereolytic activity and protein content were assayed and lyophilized cutinase preparations characterized[25][START_REF] Almeida | Flow injection analysis system for on-line cutinase activity assay[END_REF][START_REF] Sambrook | Molecular cloning, a laboratory manual[END_REF].The protein concentration was determined by the method of PEARCE (BCA assay). Protein concentrations were determined with reference to a standard, the Bovine Serum Albumin (BSA) (Merck)[25].The cutinase estereolytic activity was assayed using a spectrophotometric method, based on monitoring the hydrolysis of p-nitrophenylbutyrate (p-NPB) to nitrophenol (p-NP), a yellow compound easily identify and quantify by the absorbance at 400 nm. An aliquot (20 µl) was added to a reaction mixture composed of 980 µl of a solution in 50 mM potassium phosphate buffer pH = 7.0 also containing 0.56 mM p-NPB, 11.3 mM sodium cholate and 0.43 M tetrahydrofuran.One unit of cutinase estereolytic activity was defined as the amount of enzyme required to convert 1µmol of p-NPB to p-NP per 1 min, under controlled adjusted condition. The extinction coefficient of p-NP was considered to be 1.84 × 10 4 (M -

=

  [integration area of ester][integration area of ester] + [integration area of acid]% esterification = [RCOOR´] [RCOOH] 0 = [integration area of ester][integration area of ester] + [integration area of acid] . Unless stated otherwise, a typical reaction for ethyl ester synthesis was carried out in 7 ml working volume inside of 10 ml capped flasks with rubber stoppers (EPDM stoppers, black, Sigma-Aldrich, Germany), as to minimize evaporation or loss of volatile compounds. Alcohol, acid and n-decane were mixed thoroughly in iso-octane before addition into a new reactor containing the enzyme (lyophilized form). Initial conditions were set as to minimize enzyme inhibition by the substrates. The first sample (zero point of the reaction) was collected before enzyme addition. The enzymatic ester synthesis was performed at 30ºC in a multi magnetic stirrer at 300 rpm (S.B.S. Instruments A-23). This device enables operation of multiple parallel experiments by simultaneous use of 6 to 12 reactors. At least one blank without enzyme running in parallel for each ester synthesis reaction was performed. Experiments were carried on in duplicate leading to highly reproducible conversion results with errors lower than 5%.

  oleate) was determined by a Hewllet-Packard model 5890 gas chromatography equipped with a flame ionization detector (FID). The WCOT Fused Silica coating CP Chirasil-Dex CB column, 25m x 0.25mm, DF=0.25 (Varian Inc.) was used. N-decane was used as the internal standard and to calculate ethyl esters and respective substrates concentrations in the reaction media based on the results of GC analysis according the following specific conditions applied for each particular ethyl ester and respective substrates:

1 .

 1 Influence of chain length of acids and alcohols on enzymatic esterification 3.1.1

  systems, while hexanoic acid (C 6 ) is the shortest acid that

  while ethanol can be found in the water phase as well as in the droplets (Table

  the chain length of acids and they were found to be 10, 20 and 40 min for octanoic, decanoic and oleic acids, respectively (Figure5).

23 [ 12 ]

 2312 chemistry, furthermore miniemulsions show to be an excellent reaction medium for esterification reaction without enzyme inhibition. Ross, N.W., Schneider, H., Activities of Candida rugosa lipase and other esterolytic enzymes coated on glass beads and suspended in substrate and water vapor enzymes in thin liquid films. Enzyme MicrobTechnol. 1991, 13, 370-377.

Fig. 1 .

 1 Figure Captions

Fig. 2 . 1 HFig. 3 .

 213 Fig.2. 1 H NMR spectrum for synthesis of ethyl decanoate

Fig. 4 .

 4 Fig.4. Conversion yields at equilibrium (8h) for the synthesis of ethyl-and hexyl esters for carboxylic acids from C 6 to C 18 in miniemulsion system.

Fig. 5 .

 5 Fig. 5. The influence of the acid chain length on the synthesis parameters v and lag time of ethyl esters by cutinase in iso-octane.

Fig 6 .

 6 Fig 6. Conversion yields (8h) for the synthesis of ethyl esters by cutinase in isooctane.

Fig 6 .

 6 Fig 6. Conversion yields for the synthesis of ethyl esters by cutinase in iso-octane. 254x190mm (72 x 72 DPI)

28 Table 1 .

 281 Equimolar concentration of substrates in miniemulsion system for the synthesis of ethyl-and hexyl esters

		Biotechnology Journal	Page 28 of 36
	Acids	For ethyl esters	For hexyl esters
		(M)	(M)
	Hexanoic	1,0	0.75
	Heptanoic	0.9	0.70
	Octanoic	0.8	0.65
	Decanoic	0.7	0.6
	Oleic	0.5 F o r	0.4
		P
		e
			e r
			R
			e
			v i e
			w
			29
			Wiley-VCH

Table 2 .

 2 The composition of the miniemulsion systems with 80% of amount of water as esterification reaction medium.

	Page 29 of 36
	Ingredients	% (w/w)
	Substrates	16.56
	Hexadecane	0.66
	H 2 O	81.16
	Lutensol	1.62
	Σ	100

Table 3 .

 3 Solubility of carboxylic acids in water at 20 ºC.[START_REF]cooperation between the International Programme on Chemical Safety and the Commission of the European Communities © IPCS, CEC[END_REF]

		Page 30 of 36
	Acids	Solubility, g/100ml
	Hexanoic acid	1.1
	Heptanoic acid 0.24
	Octanoic acid	0.068
	Decanoic acid	very low
	Oleic acid	very low

Wiley-VCH Biotechnology Journal

Acknowledgements

COST Action: COST-STSM-865-02962 Fundação para a Ciência e Tecnologia (F.C.T.) for Ph.D fellowship SFRH/BD/24703/2005

Conflict of interest statement

The authors have declared no conflict of interest


Prof Carl-Fredrik Mandenius

Klaus Graumann

Torsten W Schultz

Andreas Premstaller

Ing-Marie Olsson

Emma Petiot

Christoph Clemens

Mats Welin

QbD) for biotechnology-related pharmaceuticals

Keywords: Design-of-Experiments, Design space, On-line monitoring and control, Biopharmaceuticals, Active pharmaceutical ingredients CPP, critical process parameters, CQA, critical quality attribute, DoE, multivariate analysis, NIR, near-infrared, NME, new molecular entities, PAR, proven acceptable range, PAT, process analytical technology, QbD, quality by design, SPC, statistical process control, TPP, target

. The aim of the workshop was to provide an update on the present status of using QbD in biotechnology-related applications in the pharmaceutical industry. The report summarizes the essential parts of the presentations and covers the industrial, academic, and regulatory aspects of QbD. It concludes with recommendations for further work and development.

Introduction

The guiding principle of Quality-by-Design is that quality aspects must be integrated in the early stages of the production development, since good quality and production efficiency cannot be tested into products but must be built in by design.

Quality-by-Design (QbD) and the supporting enabling technology of Process Analytical Technology (PAT) unite modern scientific methodologies in modeling, sensor technology, analytical techniques and design. The concept is well-established in the academic community, and is already successfully pursued and applied in the pharmaceutical industry in a number of ways.

QbD was initiated in the pharmaceutical industry, the national regulatory authorities, and the academic world as a means of creating an early understanding of the design alternatives available during the development of a new drug. QbD should increase competitiveness where time constraints and increased customer quality demands are significant. These goals are explained in a variety of guideline documents from national bodies and worldwide industrial organizations, e.g. the ICH quality guidelines [START_REF]Document ICH Q8 Pharmaceutical Development[END_REF][START_REF]Document ICH Q9 Quality Risk Management[END_REF][3][4]. The United States' Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) have adopted these guidelines in their regulatory framework for drug development and production [START_REF]Guidance for industry, process analytical technology, a framework for innovative pharmaceutical development, manufacture and quality assurance[END_REF].

The basics of QbD are illustrated in Figure 1. The critical quality attributes (CQAs) are the properties of the product that characterize its quality; they must be guaranteed in manufacture, otherwise the product must be discarded. Typical examples of such attributes are purity, stability, solubility, and product integrity, but ease of analysis is not uniform. The CQAs are a result of the product itself, but are also highly dependent on how the product is manufactured. This is controlled by the critical process parameters (CPPs). If the CPPs are properly selected and tuned, the right CQAs will be achieved. These must be maintained over time, a non-trivial task for biological processes given the natural variations in such systems and the time-dependent behavior of most batch operations. The design space is the region where the parameter values can lie, while the control space defines the limits for their control. The design space is the multidimensional combination and interaction of input variables (e.g. material attributes) and process parameters that have been demonstrated to provide assurance of quality. The control space is the subsection of the design space where the manufacturer chooses to operate. To do this successfully, a detailed and time-dependent understanding is essential. This is where science can contribute significantly. To accomplish this, we need reliable process analytical tools.

PAT covers those methods that are useful for designing, analyzing, and controlling manufacturing through timely measurements (i.e. during processing) of critical quality and performance attributes of raw and in-process materials and processes, with the goal of ensuring final product quality. The term analytical is viewed broadly, and includes chemical, physical, microbiological, and mathematical aspects, as well as risk analysis.

Typical PAT tools are those that enable scientific, risk-managed pharmaceutical development, manufacture, and quality assurance. QbD represents a holistic approach to the development of biotechnology-related pharmaceuticals. The novelty of the concept is evident in Table 1 and2, which compares the traditional approach with the more advanced QbD approach.

Generally speaking, QbD encourages enhancement of the manufacturer's process understanding throughout the design space versus the traditional approach of filing commitments. When fully implemented, it could lead to more science-based validation and quality testing, and could also facilitate innovation and improvements in wellunderstood processes. These prospects may be attractive to the industry not only from a regulatory and quality perspective, but in the end also economically.

In the lifecycle of a product, QbD starts with the definition of the desired product properties. Then, during the process development phase, a multitude of input variables are linked systematically with output variables. Identification of the critical variables and parameters which have an impact on quality attributes (i.e. the CQAs) takes place at this stage. In the interest of reproducibility and process economics, those variables and parameters that have an impact on process robustness should also be identified and addressed thoroughly. At the same time, an appropriate process and product control strategy leading to a final drug product with pre-determined specifications should be developed.

As pointed out above, the final state of the product, the target product profile (TPP), must be defined before process development for clinical supplies can begin. Exhaustive physiochemical and bioanalytical characterization applying state-of-the-art methods in the field of protein characterization are vital, in order to create a comprehensive picture of the product and lower the risk of failures during the clinical development stage.

Information on the composition of amino acid sequence variants, the secondary and tertiary structure, and post-translational modifications in detail; all this sets the stage for the design of expression systems, cell line screening, and process development. For example, the distribution of glycoforms of a given product may vary within mammalian cell populations, and can influence the biological properties dramatically. Careful 7 screening and analysis are required to select a productive clone which also delivers the potential for a pre-defined target profile of glyco-variants. Together with receptor-target binding data and cell-based activity assay data, sourced from actual measurements or the public domain, the TPP can then be defined and targeted process development can begin.

In the case of innovative products, including new molecular entities (NME), the QbD definition process is of a more iterative nature, and is primarily based on the desired biological properties. Factors such as available platforms for the production of the active molecule, serum half-life, formulation, aggregation propensity, and immunogenicity must be taken into consideration during development.

From this work, the CQAs of the product can be defined. In particular, the elucidation of structure-function relationships is a key aspect in the identification of CQAs, regardless of whether innovative or biosimilar products are being developed. For the process and production development, the most important question is that of which process parameters impact CQAs, and to which degree and how controllably. A risk assessment may capture the likelihood of occurrence and the potential effects of certain process parameters on process robustness and, above all, on quality. A thorough risk assessment will therefore be an integral part of this whole process.

By definition, CPPs have an impact on quality attributes, and so must be controlled tightly. Furthermore, the ICH Q8 guidelines propose a control strategy that ensures the TPP specification is reached in a reproducible manner [START_REF]Document ICH Q8 Pharmaceutical Development[END_REF]. The control strategy will be focused on (the critical) sources of variability, such as certain raw materials, but also the inherent biological processes as a whole. The control strategy therefore encompasses all input materials, unit operations, in-process testing (off-line, at-line, or on-line), and finally the release tests. The level of process understanding strongly influences the design of such a control strategy.

During development, the analytical results recurrently contribute to adaptation and optimization of the production system, leading to continuous improvement and refinement of both the process and the product specifications. PAT-based development tools, such as Design-of-Experiments (DoE), mathematical modeling, and multivariate data analysis (MVA), when carefully applied during the characterization, will create a 8 knowledge space which hosts the design space for the given process. When justified properly, movement within this design space would by definition not represent a change in the regulatory sense, and hence flexibility is increased.

Industrial applications of QbD

Biopharmaceutical companies may benefit substantially from applying QbD. One example is Sandoz Biopharmaceuticals, a leader in biosimilar product development, which has readily embraced many of the QbD elements. Based on the ICH guidelines, concept papers and development manuals have been established. Furthermore, PATrelated methods and tools such as DoE, MVA, and online monitoring tools have been adapted to in-house requirements.

MVA is currently being established on a manufacturing scale for the fermentation, isolation, and purification processes of one of Sandoz' commercial biotech products with the aim of learning more about process variability and thus finding out where to improve robustness. As a result, a QbD design space will be created which could justify regulatory flexibility. Important issues associated with this task are the inhomogeneities of signal traces due to spikes, and the generally much lower number of batches used for biopharmaceuticals in comparison to pharmaceutical products.

A number of spectroscopic tools are being developed in order to improve the control options for biological processes. One example is PTR mass spectrometry (Ionimed Analytik GmbH, Innsbruck, Austria) for assessment of patterns of volatile fermentation metabolites in real-time, thereby giving more insight into process performance and, ultimately, better process robustness.

For a company like Sandoz, QbD means a substantial investment in technologies, strategies, and skilled people. Implementation of QbD is, no doubt, a long-term undertaking and requires resources not only in technical development but also in production, quality, and regulatory support functions (see Table 2). Such investment should ultimately pay off by reducing production costs, by a more consistent quality, and by providing a more profound, science-based insight into biopharmaceutical processes. Another company involved in QbD is Boehringer Ingelheim Pharma (BIP) and its Biopharmaceutical Division. BIP is a well-established contact manufacturer in the industrial biotechnology sector for over 25 years, and is known as a developer of highly innovative drugs. For BIP it is of utmost importance to keep up with the latest technologies, developments, and regulatory requirements in order to reduce both the cost of goods and the time to market. One of the prevailing aims is to develop production processes that reproducibly yield biopharmaceutical products with high product quality. Therefore, BIP is currently pursuing the implementation of QbD concepts, including PAT as a tool to improve process understanding, as proposed by the ICH Q8 guideline [START_REF]Document ICH Q8 Pharmaceutical Development[END_REF]. An example of the implementation of a PAT tool used at BIP is described below.

Enabling PAT tools

The main enabling PAT tools of QbD, when applied to biopharmaceutical processes, consist of (1) chemometrics computation methods and (2) bioanalytical methods and devices. Below the basics are described for applying chemometrics to QbD followed by a few examples of novel applications of bioanalysis.

Chemometrics for QbD

Design-of-Experiments (DoE) [START_REF] Eriksson | Design of Experiments: Principles and Applications[END_REF][START_REF] Mandenius | Review: Bioprocess optimization using DoE methodology[END_REF] has lately experienced a revival in the pharmaceutical industry due to initiatives towards QbD. The key factor in QbD is knowledge of the process, and in general mechanistic models are seen as the final aim and proof of complete understanding. DoE is, properly applied, a powerful tool both in the development of knowledge and in the determination of mechanistic models.

In short, one can say that to gain a higher level of knowledge, experiments are necessary.

By planning the experiments according to DoE, the information gained from each experiment is maximized, thus increasing the prospects for solving the problems addressed. Using DoE as a basis for collecting information enables the calculation of predictive mathematical models that describe the relationships between changes in experimental conditions and the outcome of the experiments. Depending on the type of 10 design applied, models with varying complexity -linear, interaction, or quadratic effects -can be estimated. In addition, the mathematical model enables visualization of the functions in the system.

The importance of a properly performed problem formulation prior to DoE increases with the complexity of the investigated system. In biological systems there are many factors to consider, and in addition there are many sources of "noise" in the data due to uncontrollable factors and biological variation. It is therefore very important to perform the experiments in the DoE under as identical conditions as possible. This, in addition to the costs, makes it cumbersome to perform DoE in large scale fermentation. On the other hand, we have seen great success and gained much value and knowledge from applying DoE in small scale fermentors run in parallel with a large-scale fermentor using the same batch of nutrient feed.

Once the DoE has been finalized, the data analysis remains. The length of the fermentation processes means that sophisticated tools for data analysis are required, such as MVA, which allows analysis of process evolution. With DoE and MVA in combination a process can be characterized, the main drivers identified, and a deeper understanding gained. Modeling of the process evolution allows real-time visualization of the process status at any chosen time (Figure 2). The purpose of this could be monitoring of how the process evolves compared to a defined "golden batch" or design space, early detection of deviations, and identification of the cause of deviation. One value of these models is that they generally detect drifts earlier than classical statistical process control (SPC) charts, since multivariate models also identify deviations due to changes in correlation pattern in the process.

Bioprocess monitoring and control tools

For animal cell culture processes in particular, PAT has been considered important for improving efficiency and ensuring final product quality. To achieve these objectives, new tools for on-line culture monitoring are necessary. While pH, temperature, dissolved oxygen level, and cell density could be monitored today by in-situ probes, the components of the cell culture supernatant are still quantified by time-consuming and expensive off-line analyses. Spectroscopic methods, such as near infrared (NIR) and Raman spectroscopy, have emerged for use in following nutrient consumption or byproduct production in animal cell processes. Their advantages are various: noninvasive and non-destructive techniques, no sampling, high frequency of spectra acquisition, and large number of molecules potentially quantified. Their limitations mainly concern analysis calibration, chemometric processing of the spectra, and previous identification of strategic operating parameters and set-points. Papers reporting data on this subject are focused on off-line [START_REF] Arnold | In-situ near infrared spectroscopy to monitor key analytes in mammalian cell cultivation[END_REF][START_REF] Ren | Comparison of multivariate calibration models for glucose, urea, and lactate from near-infrared and Raman spectra[END_REF], on-line [START_REF] Rhiel | On-line monitoring of human prostate cancer cells in a perfusion rotating wall vessel by near-infrared spectroscopy[END_REF], or in situ [START_REF] Riley | Simultaneous measurement of 19 components in serum-containing animal cell culture media by Fourier transform near-infrared spectroscopy[END_REF][START_REF] Riley | Simultaneous measurement of glucose and glutamine in insect cell culture media by near infrared spectroscopy[END_REF] NIR analyses, mainly performed with suspension cell culture of CHO or Sf-9 cell lines. Ren and Arnold [START_REF] Ren | Comparison of multivariate calibration models for glucose, urea, and lactate from near-infrared and Raman spectra[END_REF] propose a comparison of multivariate calibration models for synthetic component mixtures from NIR and Raman spectra. To our knowledge, no paper has reported data with adherent cells cultivated in stirred reactors.

Proper evaluation of NIR and Raman spectroscopy methods is highly desirable for cell cultures. One example is adherent VERO cells cultivated in serum-free medium and on microcarriers in spinner flasks and stirred bioreactors (CNRS, Université-Nancy, France).

The quantification of lactate and glucose was performed on culture media samples by offline measurements, and compared to data from reference enzymatic methods. NIR spectroscopy data from chemometric analyses of spectra could be correlated to reference results, contrary to the Raman results. Furthermore, no influence of microcarriers on profile and quality of acquired NIR data was noticed, while Raman spectroscopy presented an interesting sensitivity to cell physiological state. In a second experiment, in 13 situ analyses were performed with a transflectance sterilizable NIR probe. Good correlations were observed between NIR data and off-line reference results for glucose, lactate, ammonium ions, and lactate dehydrogenase in culture medium. Thus, spectroscopy methods represent a valuable asset for adherent animal cell cultures analysis, and further developments would make these potential PAT tools for in-situ monitoring and controlling animal cell processes.

Another example of PAT is off-gas analysis, which can serve as an adequate tool to gain comprehensive information about biopharmaceutical process conditions. Traditionally, in-process control parameters have mainly included parameters such as viable cell count, viability, product concentration, glucose concentration, glutamine concentration, lactate concentration, ammonia concentration, osmolality, and pO 2 , among others. While these are valuable for the monitoring of a biopharmaceutical fermentation process, it is likely that additional physiological or metabolic data might reveal additional useful information, thus deepening the understanding of the process. One such extra key parameter could be the oxygen uptake rate (OUR). In mammalian cell cultivation, oxygen is required to produce energy for both cell growth and energy maintenance. For example, the aerobic degradation of carbohydrates (e.g. glucose) by glycolysis and the respiratory chain reaction in the mitochondrion both depend on oxygen.

Ideally, the conversion of 1 mol glucose and 6 mol O 2 yields 6 mol CO 2 , biomass and energy. Thus, a strong relationship between glucose, oxygen consumption, and carbon dioxide production is obvious with respect to the cell's energy metabolism.

While traditional approaches to measure the OUR require a balance on the liquid phase and further knowledge about the mass transfer coefficient k L a, which in turn is dependent on many parameters such as media composition, gassing system, or bubble size [START_REF] Schumpe | Gas solubilities in microbial culture media[END_REF], it is now possible to apply a mass spectrometer as a sensitive and robust method to determine the OUR in situ [START_REF] Oezemre | Measurement of oxygen uptake and carbon dioxide production rates of mammalian cells using membrane mass spectrometry[END_REF].

At Boehringer Ingelheim, the OUR of different cell culture processes was studied by using an off-gas quadrupole mass spectrometer. Online OUR data were presented together with typical culture parameters, such as product concentration, from representative fed-batch fermentations of different industrial relevant scales. It turned out 14 that the application of the off-gas quadrupole mass spectrometer with online OUR data revealed distinguishable levels of output parameters, while traditional in-process control parameters were unable to provide proof of different process characteristics.

Consequently, mass spectrometry offers a useful and precise PAT technique for gaining further insight into the cell metabolism, as well as further opportunities to monitor mammalian cell culture fermentations based on their respiratory activity.

Several other methods useful for on-line or at-line monitoring of biopharmaceutical processes have been presented and applied on a small scale [START_REF] Lantz | Determination of cell mass and polymyxin using multi-wavelength fluorescence[END_REF]. Multi-wavelength fluorimetry seems advantageous from several aspects -it is non-invasive and very sensitive; and, due to its principle, it discriminates those compounds that fit the excitation-emission combinations scanned for. However, it requires fluorescent properties of the analytes probed for. Such analyte examples are found among the antibiotic molecules and other small molecular metabolites [START_REF] Clementschitsch | Improvement of bioprocess monitoring, development of novel concepts[END_REF].

There is no doubt that analytical applications of genomic, proteomic, and transcriptomic methods provide powerful tools to PAT. The omics tools have a unique possibility to relate quality aspects to the CPP through a better understanding of systems biology aspects in biopharmaceutical production [START_REF] Duerrschmid | Monitoring of transcriptome and proteome profiles to investigate the cellular response of E. coli towards recombinant protein expression under defined chemostat conditions[END_REF]. So far, relatively few reports are available on how identification and reduction of metabolic/physiological bottlenecks of the biological systems can influence the quality of the production, and how this understanding can subsequently be exploited with QbD methodology.

Regulatory aspects on the introduction of QbD for biopharmaceuticals

Regulatory considerations for biological molecules as pharmaceutical products

The viewpoints reported above have to be in perfect compliance with the long term regulators' requirements on the biotechnology-related pharmaceuticals. Especially biological processes and starting materials are more prone to variation, and the molecules are often large and may be conjugated, for example as glycosylated and pegylated forms of the product. These may be sensitive to oxidization, deamidation, and aggregation/ fragmentation. Changes may influence activity, but changes in for example glycosylation 15 may in particular affect immunogenicity or clearance. No single method will allow for a full characterization of the product, and subtle changes may even go unnoticed [START_REF]). Document ICH Q6B Test Procedures and Acceptance Criteria for Biotechnological/Biological Products[END_REF]. At the same time, it is of key importance that the material produced on a commercial scale for the market is representative in all important aspects of what has been shown to be efficacious and safe during preclinical and clinical trials.

The old paradigm for biopharmaceuticals -"one process -one product" -could be interpreted as meaning that all changes to a production process require new clinical studies.

However, provided that comparability between the pre-and post-change product has been shown, it is possible to introduce changes to the manufacture without additional clinical studies [START_REF]Mandate for the EMEA PAT team[END_REF].

Alternative manufacturing processes (e.g. use of different clones expressing the same protein or use of significantly different purification processes) are not allowed, but flexibility within a given process is allowed if validated. The introduction of a properly validated and documented QbD system and design space in the manufacture of biological medicinal products therefore meets no formal obstacles. It should be noted that introduction of QbD is optional, and the classical way with more emphasis on end product testing will still be accepted, if appropriately documented [4].

Many aspects interact in assuring full control of the product to be marketed. Control of the raw and starting material (including cell banks) is particularly important for a biological product, where the biological origin of the material can be expected to introduce variability. In-process controls are performed to monitor whether the process is behaving as expected. The process should be validated and shown to be robust and reproducible. The production should be performed under Good Manufacturing Practices (GMP), in order to ensure reliability, to assure adherence to approved production methods, and to avoid the risk of contamination.

As a final part of the quality control comes the testing of the active substance and the finished medicinal product. These tests do not cover all aspects of the product, but a number of relevant tests are picked to mirror the product characteristics under normal production conditions. From these, and by taking risk assessment aspects into account, the control strategy is formed. All parts need to be included, but the focus put on each of 16 them may differ. When there is enhanced process understanding and appropriate upstream testing and monitoring, testing of the finished stages may be reduced [START_REF]Document ICH Q8 Pharmaceutical Development[END_REF].

In most cases the manufacturing process is changed during development, and as mentioned above the applicant needs to show comparability throughout the whole development in order to be able to refer to data gathered from earlier versions of the process [START_REF]Mandate for the EMEA PAT team[END_REF]. It is also expected that if a design space is established in the process, much in-depth characterization will be required to verify that all combinations of CPP within the design space will result in a comparable outcome, in terms of CQAs, to what has been shown in clinical trials to be safe and efficacious.

Difficulties may arise relating to the fact that only a few batches are used in clinical trials, and hence these may not cover the full variability that can be expected in regular production. An enhanced understanding of structure-effect relationship may therefore simplify the establishment of models and allow the ruling out of non-significant differences.

For a deeper understanding of the processes, it is also important to evaluate potential interactions between process parameters; by multivariate analysis or other methods [4].

Aspects to consider in the introduction of QbD-based approaches

In the cases when similar molecules are produced, for example, monoclonal antibodies, platform technologies are often used. However, prediction models have been shown to be invalidated by small differences in, for example, buffers. To be fully able to extrapolate from one product to another, it is important that the model is shown to also be fully relevant to products for which the platform was not initially established.

According to the control strategy concept, the sole fulfillment of a specification cannot be considered a design space, since end testing is only one part of the strategy and the specification will be process specific. Deviations from the accepted process may result in impurities that will go unnoticed, as the specification is not designed to cover them. 17 Furthermore, a combination of proven acceptable ranges (PARs) established by varying one parameter at a time while keeping everything else locked will not be considered a design space unless it has been shown that the parameters do not interact.

When the QbD principles are introduced, the objective may not only include improved quality, fewer rejections, and continuous production optimization, but also the possibility of reduction in end product quality control testing or a reduction in filing of variations to the Market Authorization. This will be considered acceptable, provided that at the end of the manufacturing process there is an assurance of a product of predefined quality, linked to what has been used to verify safety and efficacy [START_REF]Document ICH Q8 Pharmaceutical Development[END_REF]. As monitoring and quality decisions will mainly be based on the parameters/attributes that are considered critical, the arguments for classifying something as critical or non-critical need to be wellexplained to allow for an assessment of the control strategy.

The new concept will have an impact on the work of regulators. The basis for approval will not only be assessment of documentation submitted in the MAA, but also documentation considered during inspections and assessment of the quality systems [3] and risk assessment strategies [START_REF]Document ICH Q9 Quality Risk Management[END_REF] in place. This requires a close collaboration between assessors and inspectors. To help with this and to keep up with the evolution of the field, a specialized group, the EMEA PAT team, has been established. This group consists of members from the Quality Working Party, the Biologicals Working Party, and the ad hoc Inspectors' Working Party. It has regular contact with industry organizations and other regulatory agencies to discuss common issues and provide expert knowledge for applications dealing with QbD/PAT issues. Reports and question-and-answer sessions are published on the EMEA website [START_REF]Reflection paper on PAT related information[END_REF][21][22].

As pointed out above, it is vital to make the link to the processes used to show safety and efficacy in clinical trials. All flexibility introduced and all changes made to production must result in a comparable product in all important aspects; relaxation of the level of assurance in this respect compared to the current situation will not be accepted. A change in the control strategy (e.g. less end product testing, increased flexibility within an approved design space) is however allowed for, as long as equal or better quality can be assured. Good transparency is of major importance for the successful introduction of While industry and regulators agree on the principles, interpretation differs between the two and also within the industry and regulator groups themselves. QbD can be introduced in many different ways, but it is important to agree on the basic principles and definitions in order to avoid misunderstanding. The ICH Implementation working group will have an important task in this respect.

Recommendations

The workshop resulted in a number of recommendations for how QbD in the area of biotechnology-related pharmaceuticals can be improved and facilitated. The recommendations are far from comprehensive, but are intended to highlight a few pertinent issues. The following issues appear to be of particular urgency and relevance: i) Better data management tools. Biotechnology-related manufacturing generates a large volume of data, which may be relevant or irrelevant to QbD analysis. This large volume, along with the complex structure of the data, means that it is difficult to make an overview, and so efficient analysis becomes unrealistic. Further development of knowledge management systems [3], including more powerful data management software tools, would be a valuable asset in mining for useful information.

ii) Retrieval of historical data. When it comes to the use of historical data, one hurdle is the availability of data. It is commonly stored for storing instead for stored for use, meaning that it is difficult to retrieve and organize for multivariate modeling. This issue has been recognized and addressed by many providers of data storage systems. It should also be remembered that in most cases historical data does not contain enough variability to yield all valuable information about a process. For existing processes, historical data is usually the first step in a QbD investigation, but to fully explore the process a DoE is required. The design space and control space as defined in QbD

Figure 1 .

 1 Figure 1. The design space and control space as defined in QbD

2

  Adapting industrial production systems and procedures for QbD

  Manuscript for Biotechnology Journal (Wiley): Special issue from ESBES-7 6

  Manuscript for Biotechnology Journal (Wiley): Special issue from ESBES-7

  Manuscript for Biotechnology Journal (Wiley): Special issue from ESBES-7 9

  Manuscript for Biotechnology Journal (Wiley): Special issue from ESBES-7

Figure 2 .

 2 Figure 2. By combining DoE and multivariate data analysis techniques, a multi step process can be summarized in one overview model. This model links the entire history of a lot or batch to its final properties.

  Manuscript for Biotechnology Journal (Wiley): Special issue from ESBES-7

  Manuscript for Biotechnology Journal (Wiley): Special issue from ESBES-7

  Manuscript for Biotechnology Journal (Wiley): Special issue from ESBES-7

  Manuscript for Biotechnology Journal (Wiley): Special issue from ESBES-7

  The rationale for the proposals must be clearly described and supported by documentation that describes the establishment of the proposed process in sufficient detail. Raw data should not normally be submitted, but further information may be requested if needed.

  Manuscript for Biotechnology Journal (Wiley): Special issue from ESBES-7[START_REF]Mandate for the EMEA PAT team[END_REF] iii) Process understanding for control. Ultimately, enhanced process understanding in combination with an appropriate control strategy could lead to real-time release. This long-term goal for biopharmaceuticals will certainly require better insight into what is currently often considered a black box. The following applied research topics could significantly improve our understanding of biological processes: (1) systems biology studies with omics tools to better understand metabolic networks and responses to stress and environmental conditions in general, (2) engineering of currently used hosts to eliminate bottlenecks, leading to a toolbox of optimized expression systems, (3) defined cultivation media, (4) understanding of structure-function relationships of the target molecule, and (5) improved downstream processing steps and purification concepts. iv) Criticality analysis and risk management. The parameters in bioprocess engineering mostly differ from those for chemical drug substances. Consequently, they are of different origin and have a different impact on risk. Thus, a critical analysis of parameters, occurrence, and measurability would facilitate prioritization of specific needs.v) New real-time or just-in-time biospecific analyses and assays.There is a need for further development of novel on-line/at-line monitoring tools for process development, and novel on-line/at-line monitoring tools for manufacturing. This should include rapid bio-specific analytical methods -such as glycoforms of proteins, other product variants, and side-product analysis in biotech process steps -which are not easily covered with typical systems biology analysis but call for more tailor-made assays. Biochemical engineering could contribute to further development of such methods on the basis of existing bio-recognition principles (e.g. biosensors, advanced multidimensional analyzers).Manuscript for Biotechnology Journal (Wiley): Special issue from ESBES-7 24

Figure 1 .

 1 Figure 1.

Figure 2 . 25 Figure 1 Figure 1 . 26 Figure 2

 22511262 Figure 2.

Figure 2 .

 2 Figure 2. By combining DoE and multivariate data analysis techniques, a multi step process can be summarized in one overview model. This model links the entire history of a lot or batch to its final properties.

Table 1 Comparison of the traditional and QbD approaches Table 2 Important features of the QbD vision

 1

	Regulatory scrutiny adjusted to level of
	Process Understanding.

Continuous improvement allowed within Design Space.

		Changes managed within company's
		quality system. Real time batch release
		feasible. Higher
	Regulatory	Compliance focus.
		Changes require prior approval,
		lengthy process, uncertain
		outcome.

reliance / trust / understanding on systems.

			Quality decisions and filing
			commitments based
	Quality	Post-manufacture sampling and	Management of variability.
		quality testing.	Process control focused on critical
		Process Validation.	attributes.
			Continuous Quality Verification.
	Systems	Systems designed to inhibit
		changes & minimize business risks.
		Discourages improvement &
		innovation.

on Process Understanding and Risk Management. Design Space concept.

		Traditional Approach	QbD Approach
	Broad	Quality decisions 'divorced' from
	Concept	science and risk evaluation.
		Adherence to filing commitments.
			Regulatory scrutiny adjusted to level of
			Process Understanding.

Continuous improvement allowed within Design Space.

		Changes managed within company's
		quality system. Real time batch release
		feasible.
	Regulatory	Compliance focus.
		Changes require prior approval,
		lengthy process, uncertain
		outcome.

Higher reliance / trust / understanding on systems.

		Management of variability.
		Process control focused on critical
		attributes.
	Systems	Systems designed to inhibit
		changes & minimize business risks.
		Discourages improvement &
		innovation.

Continuous Quality Verification.

	Post-manufacture sampling and
	quality testing.
	Process Validation.

Quality Quality decisions and filing commitments based on Process Understanding and Risk Management. Design Space concept.

	Quality decisions 'divorced' from
	science and risk evaluation.
	Adherence to filing commitments.

Broad Concept QbD Approach Traditional Approach

	Source: Adapted from EFPIA, PAT Topic Group, 2005
		Starting point (Minimal approach)
		• mainly empirical • one variable at a time	• systematic, relating input material attributes, process parameters to critical quality attributes • multivariate experiments • establishment of a
	Manufacturing process	• fixed • focus on reproducibility • validation based on full-scale batches	• adjustable within design space • focus on control strategy and robustness lifecycle approach to validation, continuous process verification
	Process controls	• in-process tests primarily for go/no-go decisions • off-line analysis	• PAT tools with feed forward and feedback loops • use of statistical process control methods • process ops tracked to support continual improvement efforts post-approval
	Control strategy	• Quality controlled primarily by intermediate and end product testing	• Quality ensured by a risk-based control strategy for a well understood product and process Quality controls shifted upstream; real time release or reduced end-product testing
	Product specifications	• Primary means of control • Based on batch data available at time of registration	• Part of the overall quality control strategy • Based on desired product performance with relevant supportive data
	Lifecycle management	• Reactive (problem solving, corrective action)	• Preventive action • Continual improvement facilitated

design space Final QbD approach (Vision) Development Aspect

  Manuscript for Biotechnology Journal (Wiley): Special issue from ESBES-7

		F o
		r
		P
		e e r
	controls Process Manufacturing process Development Aspect	• PAT tools with feed forward and feedback loops • use of statistical process control methods • process ops tracked to support continual improvement efforts post-approval • adjustable within design space • focus on control strategy and robustness lifecycle approach to validation, continuous process verification • systematic, relating input material attributes, process parameters to critical quality attributes • multivariate experiments • establishment of a design space Final QbD approach (Vision) R • validation based on full-scale go/no-go decisions • off-line analysis • in-process tests primarily for • fixed • focus on reproducibility batches • mainly empirical • one variable at a time Starting point (Minimal approach) e v i e w
	Control strategy	• Quality controlled primarily by intermediate and end product testing	• Quality ensured by a risk-based control strategy for a well understood product and process Quality controls shifted upstream; real time release or reduced end-product testing
	Product specifications	• Primary means of control • Based on batch data available at time of registration	• Part of the overall quality control strategy • Based on desired product performance with relevant supportive data
	Lifecycle management	• Reactive (problem solving, corrective action)	• Preventive action • Continual improvement facilitated
	adapted from draft Annex to ICH guideline Q8

Wiley-VCH Biotechnology Journal

Wiley-VCH Biotechnology Journal

23