domain: chim phys

text:
Beatrice Adelizzi

Nathan J Van Zee

Lafayette N J De Windt

Anja R A Palmans

E W Meijer

The future of supramolecular copolymers unveiled by reflecting on covalent copolymerization

Supramolecular copolymers are an emerging class of materials, and in the last years their potential has been demonstrated on a broad scale. Implementing noncovalent polymers with multiple components can bring together useful features such as dynamicity and new functionalities. However, mastering and tuning the microstructure of these systems is still an open challenge. In this perspective we aim to trace the general principles of supramolecular copolymerization by analyzing them through the lens of the well-established field of covalent copolymerization. Our goal is to delineate guidelines to classify and analyze supramolecular copolymers in order to create a fruitful platform to design and investigate new multicomponent systems.

 INTRODUCTION

While one-dimensional supramolecular polymers have long been employed by Nature as a powerful structural motif, scientists have only recently begun to exploit their dynamicity, directionality, and functionality. This confluence of properties makes them promising for applications in pharmaceuticals, nanoelectronics, and catalysis, but many fundamental principles that dictate the structure of supramolecular polymers remain unresolved. In 2009, we proposed to draw inspiration for future research directions by carefully considering the maturation of its older sister, covalent polymerization. [START_REF] De Greef | Supramolecular Polymerization[END_REF] Following Carothers's classic classification, we organized supramolecular polymers based on the process through which each aggregate is formed. Connections between the two fields became strikingly clear. Isodesmic polymerization can be considered a non-covalent analogue of step growth polymerization, characterized by high polydispersity and a degree of polymerization that depends on the association constant of the supramolecular units. Ring-chain supramolecular polymerization is similar to ring opening polymerization and depends on the equilibrium between linear and cyclic aggregates. Finally, cooperative supramolecular polymerization can be correlated to chain growth polymerization. This process is typically under thermodynamic control; although kinetically trapped copolymers are reported, over time such species generally convert into the thermodynamically most stable state. These connections helped to highlight the distinctions between covalent and supramolec-ular polymerization processes, rendering underlying research needs into tangible goals.

In this context, one of the directions that we identified was the intriguing challenge to make well-defined supramolecular copolymers. At that time, in contrast to the multitude of methods available to tailor covalent copolymers, there were few synthetic strategies to control the microstructure of supramolecular copolymers. The challenge is the inherent lability of non-covalent bonds, which are typically kinetically and thermodynamically less stable than covalent bonds. However, in the past ten years, considerable inroads have been made to reconcile the dynamicity of supramolecular copolymers with the demand for microstructural control. Many studies and comprehensive reviews [START_REF] Liu | Supramolecular Chirality in Self-Assembled Systems[END_REF][START_REF] Yashima | Supramolecular Helical Systems: Helical Assemblies of Small Molecules, Foldamers, and Polymers with Chiral Amplification and Their Functions[END_REF][START_REF] Das | H-Bonding Directed Programmed Supramolecular Assembly of Naphthalene-diimide (NDI) Derivatives[END_REF][START_REF] Yang | Hydrogen Bonding Modules for Use in Supramolecular Polymers[END_REF][START_REF] Van Der Zwaag | Programmable Supramolecular Polymerizations[END_REF] have since been published focusing on nucleation-elongation processes, kinetic control, competition, pathway complexity, and supramolecular monomer design.

Given the field's nascence, we feel it is timely to critically reflect on recent progress through the lens of covalent chain copolymerization to illuminate challenges and opportunities for the future. Considering the ubiquity of well-defined covalent copolymers, we believe that controlling the structure of one-dimensional supramolecular copolymers is paramount to realize next-generation functional materials. Our objective is to generate insight into urgent research directions by creating a strong link between covalent and non-covalent copolymerization worlds. The first supramolecular copolymers, such as the one reported by Lehn and co-workers, [START_REF] Fouquey | Molecular Recognition Directed Self-assembly of Supramolecular Liquid Crystalline Polymers from Complementary Chiral Components[END_REF] are based on an isodesmic mechanism and non-covalent interactions among complementary units, but in this work we mainly focus on cooperative chain growth processes. In isodesmic (step) copolymerization, the sequence of repeating units is either dictated by differing complementarity or randomized in the case of equal reactivity. For cooperative (chain) copolymerization, the challenges are similar to those of their covalent counterparts. As a point of reference, we chose to structure this work based on the organization employed in the chain copolymerization chapter of Odian's classic textbook Principles of Polymerization. [START_REF] Odian | Principle of Polymerization[END_REF] We focus on the classification and synthesis of state-of-the-art structures, discussing the parallel roles of direct characterization and theoretical models that are fundamental for the development of the field.

 SUPRAMOLECULAR CHAIN GROWTH COPOLYMERIZATION

Covalent and non-covalent copolymerization: Similarities and differences. The mechanisms of covalent chain growth copolymerization and cooperative supramolecular (chain growth) copolymerization carry several similitudes. At the outset of both processes, a certain activation is required to initiate polymer growth. For covalent polymers, a reactive initiator (e.g., a radical, cation, or anion) reacts with a monomer to generate an active propagating species that enchains additional monomers (Figure 1a). [START_REF] Odian | Principle of Polymerization[END_REF] Similarly, supramolecular polymers require the generation of a nucleus (e.g., generally an energetically unfavorable small aggregate) to initiate the cooperative growth of a supramolecular copolymer (Figure 1b). [START_REF] De Greef | Supramolecular Polymerization[END_REF] Just as in covalent processes, the initiation and growth of supramolecular copolymers are sensitive to impurities, [START_REF] Szwarc | Molecular Weight Distribution of "Living Polymers," II. Effect of Impurities[END_REF][START_REF] Hirschberg | Supramolecular Polymers from Linear Telechelic Siloxanes with Quadruple-Hydrogen-Bonded Units[END_REF] co-solvents, [START_REF] Xu | Effect of Cosolvents on the Precipitation Polymerization of Acrylic Acid in Supercritical Carbon Dioxide[END_REF][START_REF] Korevaar | Controlling Chemical Self-Assembly by Solvent-Dependent Dynamics[END_REF][START_REF] Weng | Control of Gel Morphology and Properties of a Class of Metallo-Supramolecular Polymers by Good/Poor Solvent Environments[END_REF] reaction time, and temperature. All these considerations dictate average molecular weight and microstructure. [START_REF] Greenley | Free Radical Copolymerization Reactivity Ratios[END_REF][START_REF] Flory | Principles of Polymer Chemistry[END_REF] Just as in covalent copolymerization, changes to viscosity [START_REF] Flory | Principles of Polymer Chemistry[END_REF][START_REF] Burnworth | Optically Healable Supramolecular Polymers[END_REF][START_REF] Mark | Physical Properties of Polymers[END_REF] and optical properties [START_REF] Mark | Physical Properties of Polymers[END_REF][START_REF] Verbiest | Strong Enhancement of Nonlinear Optical Properties Through Supramolecular Chirality[END_REF][START_REF] Jelley | Spectral Absorption and Fluorescence of Dyes in the Molecular State[END_REF][START_REF] Eisfeld | The J-and H-Bands of Organic Dye Aggregates[END_REF] of a supramolecular polymer solution or melt signal the progress of reaction. Moreover, several synthetic strategies used to control structure in both covalent and supramolecular chemistries share mechanistic similarities. For example, the use of chain transfer agents and chain stoppers provides control over the average length of covalent and supramolecular polymers, respectively. [START_REF] Lortie | Chain Stopper-Assisted Characterization of Supramolecular Polymers[END_REF][START_REF] Ahlers | Dynamic Light Scattering Investigation of the Kinetics and Fidelity of Supramolecular Copolymerizations in Water[END_REF][START_REF] Smulders | Cooperative Two-Component Self-Assembly of Mono-and Ditopic Monomers[END_REF] In addition, the recent introduction of supramolecular living polymerization [START_REF] Würthner | Supramolecular Polymerization: Living It up[END_REF][START_REF] Kang | A Rational Strategy for the Realization of Chain-Growth Supramolecular Polymerization[END_REF][START_REF] Ogi | Impact of Alkyl Spacer Length on Aggregation Pathways in Kinetically Controlled Supramolecular Polymerization[END_REF][START_REF] Ogi | Mechanism of Self-Assembly Process and Seeded Supramolecular Polymerization of Perylene Bisimide Organogelator[END_REF][START_REF] Ogi | Living Supramolecular Polymerization Realized through a Biomimetic Approach[END_REF][START_REF] Endo | Photoregulated Living Supramolecular Polymerization Established by Combining Energy Landscapes of Photoisomerization and Nucleation-Elongation Processes[END_REF][START_REF] Aliprandi | Controlling and Imaging Biomimetic Self-Assembly[END_REF] has unlocked control over copolymer length and sequence analogous to living chain copolymerization.

However, the nature of the bonds that hold supramolecular and covalent polymers together are sharply distinct. In contrast to the thermodynamic stability of covalent copolymers, supramolecular copolymers are dynamic because they are held together by comparatively weak non-covalent interactions (e.g., hydrogen bonding, π-π stacking, and van der Waal's forces). The monomer sequence of a supramolecular copolymer is fluxional, as monomers can change their relative positions within a given polymer chain or integrate into other chains. Consequently, supramolecular polymers do not terminate in the way that covalent polymers do. These properties impart unique adaptability and modularity to the resulting materials, but they render the analysis of their structure a great challenge. Another striking difference is that supramolecular polymerizations often show pathway complexity. [START_REF] Korevaar | Pathway Complexity in Supramolecular Polymerization[END_REF] Small differences in the conditions of aggregation (e.g., variation in temperature or solvent) can cause a given set of monomers to form different self-assembled structures. [START_REF] Korevaar | Pathway Complexity in π-Conjugated Materials[END_REF][START_REF] Van Der Zwaag | Kinetic Analysis as a Tool to Distinguish Pathway Complexity in Molecular Assembly: An Unexpected Outcome of Structures in Competition[END_REF][START_REF] Valera | Tunable Energy Landscapes to Control Pathway Complexity in Self-Assembled N-Heterotriangulenes: Living and Seeded Supramolecular Polymerization[END_REF] Covalent polymerizations are also sensitive to changes in reaction conditions, giving rise to fluctuations in, for example, polydispersity, tacticity, and monomer sequence. However, excluding vitrimers and other reversible polymers, these changes are permanently encoded into the final covalent polymers. In the case of pathway complexity, as a consequence of the inherent dynamicity of supramolecular polymers, the concentration of each type of aggregate is not constant and responds to changes in reaction conditions. The ability to tune aggregated structures at will highlights the extreme sensitivity and potential utility of these structures. [START_REF] Ogi | Kinetic Control over Pathway Complexity in Supramolecular Polymerization through Modulating the Energy Landscape by Rational Molecular Design[END_REF] The non-innocent effect of solvents in supramolecular polymerization. Before proceeding, it is important to consider solvent in defining copolymer compositions. In the case of covalent copolymerization, the choice of solvent can be an important tool for tuning relative polymerization rates, which ultimately determines the microstructure of a copolymer. The interaction between the solvent and the propagating polymer species is frequently supramolecular in nature. For example, solvent polarity dictates the strength of ion pairing in the anionic copolymerization of vinyl monomers, which modulates the reactivity of the propagating carbanion chain-end toward different monomers depending on their respective structures. [START_REF] Odian | Principle of Polymerization[END_REF] The resulting polymers are then typically represented in terms of their polymerized monomer units. When dispersed in a solvent, the solvent interacts with the polymer to some degree depending on the temperature and the reactivities of the polymer and the solvent. However, these associated solvent molecules are generally not included in the polymer's chemical structure because they are not covalently bonded to the polymer. This distinction does not exist in the case of supramolecular copolymerization because solvent molecules interact with supramolecular copolymers via the same kinds of noncovalent forces that hold the monomers together. For this reason, a supramolecular "homopolymerization" of a single monomer may not be a homopolymerization at all. Solvent molecules could serve an essential role in supporting the fiber's supramolecular structure, and thus such a naming convention is potentially misleading because it deemphasizes the importance of solvent molecules as structural units. It is nevertheless difficult to unambiguously assign the role of solvent in most supramolecular systems, and so we acknowledge the necessity of simplifying supramolecular polymer structures by describing them primarily in terms of their principle monomer component(s).

Solvent effects have been reviewed elsewhere, [START_REF] Liu | Supramolecular Chirality in Self-Assembled Systems[END_REF][START_REF] Biedermann | Experimental Binding Energies in Supramolecular Complexes[END_REF] but here we would like to highlight recent efforts to exploit the action of solvents as a means to control supramolecular structure. The most common manifestation of this approach is to add a "good" solvent (i.e., a solvent that competitively associates with the supramolecular units) to denature supramolecular fibers, thereby controlling their average size. Aida and co-workers [START_REF] Venkata Rao | Thermally Bisignate Supramolecular Polymerization[END_REF] recently expanded this principle by balancing denaturation with the self-association of the denaturant to realize bisignate supramolecular polymerization as a function of temperature. Others have shown that the helicity of one-dimensional fibers-usually evaluated using circu-lar dichroism spectroscopy-can be controlled through tuning the interplay of structure and reactivity between both the supramolecular polymer and the solvent. Our group and others [START_REF] Kulkarni | Solvent Clathrate Driven Dynamic Stereomutation of a Supramolecular Polymer with Molecular Pockets[END_REF][START_REF] Cantekin | A Stereoselectively Deuterated Supramolecular Motif to Probe the Role of Solvent during Self-Assembly Processes[END_REF] have shown that the shape of the solvent can directly influence the helicity of supramolecular fibers, suggesting that molecular packing of solvent into the fiber's corona gives rise to perturbations to the inner core of the fiber.

Water must also be acknowledged for its importance as a structural element in supramolecular polymers in bulk and in solution. In the bulk, Qi, Schalley, and co-workers 41 demonstrated that water acts as an essential structural component that unlocks impressive adhesive properties of supramolecular polymers formed with crown etherfunctionalized benzene tricarboxamides. In aqueous media, Han, Stupp and co-workers [START_REF] Ortony | Water Dynamics from the Surface to the Interior of a Supramolecular Nanostructure[END_REF] establish a striking resemblance between the interaction of water and synthetic peptide nanofibers with the interaction between water and natural supramolecular materials such as proteins. Water in the interior of such fibers exhibit rapid diffusion, while water at the surface of the fiber is essentially immobilized, suggesting that these water molecules form hydrogen bonds with the fiber and serve an integral role in supporting the fiber's structure. In organic solvents as well, several reports [START_REF] Johnson | Molecular Basis for Water-Promoted Supramolecular Chirality Inversion in Helical Rosette Nanotubes[END_REF][START_REF] Gillissen | Triple Helix Formation in Amphiphilic Discotics: Demystifying Solvent Effects in Supramolecular Self-Assembly[END_REF][START_REF] Liu | Water Tuned the Helical Nanostructures and Supramolecular Chirality in Organogels[END_REF][START_REF] Liu | Supramolecular Helical Nanofibers Formed by an Achiral Monopyrrolotetrathiafulvalene Derivative: Water-Triggered Gelation and Chiral Evolution[END_REF][START_REF] Yu | Effect of Water on the Supramolecular Assembly and Functionality of a Naphthalimide Derivative: Tunable Honeycomb Structure with Mechanochromic Properties[END_REF][START_REF] Zee | Potential Enthalpic Energy of Water in Oils Exploited to Control Supramolecular Structure[END_REF][START_REF] Xing | Water-Binding-Mediated Gelation/Crystallization and Thermosensitive Superchirality[END_REF] point to the fundamental relationship between supramolecular structure and the presence of water-even trace amounts in highly apolar solvents. Our group [START_REF] Zee | Potential Enthalpic Energy of Water in Oils Exploited to Control Supramolecular Structure[END_REF] recently reported that the monomeric nature of water in alkanes gives rise to a potent thermodynamic driving force for it to bind to codissolved supramolecular polymers and effect profound changes to their structures. We believe that this growing body of research expresses both the complexity and import of solvent molecules as not merely spectators but as active participants in one-dimensional supramolecular polymerization.

Type of copolymers. Linear covalent copolymers are commonly categorized by the pattern of monomer enchainment. For example, for a polymer made up of monomers A and B, we can assign it as random, alternating, block, blocky, or periodic (Figure 2). Random copolymers do not have a regular pattern of monomer enchainment.

Alternating copolymers, represented as poly(A-alt-B), contain the monomers in a regular alternating pattern. A related microstructure is periodic, which has the monomers sequenced in a regular pattern; for example, poly(A2-per-B) indicates that two A monomers are enchained in a row followed by one B monomer. Block and blocky copolymers contain both long sequences of each monomers in the copolymer chain. Diblock copolymers, poly(A-b-B), are linear polymers with long sequences of each monomers, while multiblock ("blocky") copolymers exhibit multiple short runs of each monomer in a given polymer. As presented in Figure 2c, we classify supramolecular structures using the same IUPAC formalism that is used for covalent polymers.

In the next sections, we critically review the theoretical tools and synthetic strategies for constructing well-defined one-dimensional structures. We shall consider the range of known microstructures and the limits of structural characterization. We focus especially on synthetic strategies and characterization challenges.

 SUPRAMOLECULAR COPOLYMER COMPOSITION

As in covalent copolymerization, theoretical models are essential for understanding the composition of supramolecular copolymers. Theory is especially valuable here because the inherent dynamicity of supramolecular copolymers makes it difficult to experimentally determine microstructure. In the model developed by ten Eikelder and Markvoort, [START_REF] Adelizzi | Supramolecular Block Copolymers under Thermodynamic Control[END_REF] this behavior is accounted for by treating supramolecular copolymerizations as a set of reversible reactions between monomers and polymers. Similar to the terminal model of chain copolymerization, the equilibrium constant of a reaction is assumed to depend only on the identity of the last-enchained monomer; an added assumption is that monomers can only add to the end of a growing chain. For example, the co-assembly of A and B to form supramolecular copolymer P can thus be described by four reactions (Figure 3a) that provide the basis for deriving expressions for the concentration and composition of the copolymers. The concentration of copolymers is expressed in terms of the concentration of polymerized monomer units ([M]pol), which in turn can be expressed in terms of the total concentration of monomer ([M]tot) and the concentration of free monomer ([M]free, Figure 3b, top). A similar equation can be derived for the fraction of polymerized monomers φpol (Figure 3b, bottom). To determine the concentration of free monomer as well as the concentration and composition of the copolymers, the elaborated forms of these mass balance equations are solved numerically with the input consisting of the known [M]tot and the experimentally determined equilibrium constants KA-A, KA-B, KB-A, and KB-B (Figure 3a).

From this model and subsequent fitting, we can extract thermodynamic parameters that help us to understand supramolecular microstructure. As in covalent copolymerization, reactivity ratios are a useful tool for comparing microstructures when the monomers are less, equally, or more reactive in copolymerization compared to homopolymerization. Here, the reactivity ratio R is defined as the ratio of Gibbs free energy change of the homo-aggregation (ΔGA-A + ΔGB-B) to the Gibbs free energy change of the heteroaggregation (ΔGA-B + ΔGB-A, Figure 3c). For values of R greater than unity, monomers preferentially interact with their own type of monomer, while for values of R smaller than unity, the monomers tend to co-assemble.

This behavior is reflected in the shape of the copolymerization curve obtained for each class of R. As an example, we simulated the results for the temperature-induced supramolecular copolymerization of A and B. We arbitrarily set the thermodynamic parameters such that the elongation temperatures for poly(A) and poly(B) are 60 and 70 °C, respectively. We then varied the value of R by keeping the values of ΔGA-A and ΔGB-B constant and modifying the values of ΔGA-B and ΔGB-A. In this way, we modulate the tendency of the monomers to interact with each other while holding their tendency to homopolymerize constant. The resulting copolymerization curves (shades of grey), as well as the fraction of polymerized A and B (red and blue, respectively), are presented in Figure 3d. As R approaches infinity, two elongation temperatures are clearly evident, consistent with self-sorted homopolymerization of A and B. As the value of R is reduced, only one elongation temperature is evident, suggesting some degree of copolymerization. This elongation temperature shifts toward higher temperatures as the value of R is decreased-that is, the copolymer forms at higher temperature as hetero-aggregation becomes more favorable. The incorporation of A and B in poly(A-co-B) depends on R as well. A coinciding incorporation of the two monomers is visible for alternating systems (R < 1) whereas increasing the value of R results in a greater disparity between the red and blue curves. A useful extrapolation is the calculation of the fraction of polymerized A, FA, as a function of temperature (Figure 3e andf). For R > 1 (black curve), at high temperatures, the copolymers are rich in monomers of type A. As temperature decreases, monomers of type B enter into the copolymers, and the composition of the copolymers approaches the composition of the mixture of monomers. For R < 1 (lightest grey curve), where alternating structures can be formed, at high temperatures monomers B are already incorporated into the copolymers and the copolymers' composition already resembles the composition of the mixture. Thus, as the value of R decreases, monomers enter into the copolymers more by interacting with the other type of monomer and less by interacting with their own type of monomers.

An important issue to keep in mind, as with all models, is that care must be taken to evaluate the validity of the assumptions for the system at hand. This model strictly considers the interactions between two monomers in a fully reversible systems-complications due to solvent interactions, pathway complexity, and kinetic traps are excluded. In cases where such factors strongly influence self-assembly, one can either modify the model to account for new interactions or use the base model without fitting to generate simulations for qualitative comparison.

 MICROSTRUCTURE OF COPOLYMERS

Sequence-length distribution. For the sake of simplicity, the average copolymer microstructure is usually discussed. However, as indicated by the terminal model for covalent copolymerization and the thermodynamic model presented above for supramolecular copolymerization, the composition of a given copolymerization-and thus its precise microstructure-changes as a function of conversion. For covalent copolymers, small changes in microstructure can have important implications for their properties. [START_REF] Ouchi | Alternating Sequence Control for Carboxylic Acid and Hydroxy Pendant Groups by Controlled Radical Cyclopolymerization of a Divinyl Monomer Carrying a Cleavable Spacer[END_REF] This challenge was a key motivation to develop sequence-controlled copolymerization such that the monomer sequence can be unambiguously controlled throughout the copolymerization. [START_REF] Lutz | Sequence-Controlled Polymers[END_REF] Interestingly, this issue has a different meaning in the context of supramolecular copolymers because of their dynamicity. The inter-and intra-chain exchange of monomers tends to homogenize the composition for all copolymer chains in a given system. [START_REF] Albertazzi | Probing Exchange Pathways in Super-Resolution Microscopy[END_REF] Despite a growing number of strategies to achieve new supramolecular copolymer microstructures, unambiguous structural determination persists as a key challenge in supramolecular copolymerization. In this case, the monomers' exchange and the intrinsic dynamicity complicate the characterization of the microstructure. There are several outstanding reports of supramolecular copolymers that provide strong evidence for alternate, sequenced and block microstructures; we critical review them here and use them as a guide to reconsider some seminal works where the microstructure determination was not possible.

Alternating copolymers. Alternating supramolecular copolymers were the first category of supramolecular copolymers reported. In 1990 Lehn and co-workers presented seminal works on 1D poly(A-alt-B) structures based on multiple hydrogen-bonded complementary units. [START_REF] Fouquey | Molecular Recognition Directed Self-assembly of Supramolecular Liquid Crystalline Polymers from Complementary Chiral Components[END_REF][START_REF] Lehn | Perspectives in Supramolecular Chemistry -From Molecular Recognition towards Molecular Information Processing and Self-Organization[END_REF] Other examples of isodesmic copolymerization using complementary units [START_REF] Sivakova | Nucleobases as Supramolecular Motifs[END_REF][START_REF] Sijbesma | Reversible Polymers Formed from Self-Complementary Monomers Using Quadruple Hydrogen Bonding[END_REF][START_REF] Blight | AAA-DDD Triple Hydrogen Bond Complexes[END_REF] and organometallic supramolecular systems [START_REF] Gohy | Supramolecular Block Copolymer Micelles[END_REF] have since been reported by several groups. Another com-mon structural motif exploits donor-acceptor monomers that copolymerize via charge transfer interactions. [START_REF] Das | Supramolecular Assemblies by Charge-Transfer Interactions between Donor and Acceptor Chromophores[END_REF][START_REF] Jalani | Mixed Donor-Acceptor Charge-Transfer Stacks Formed via Hierarchical Self-Assembly of a Non-Covalent Amphiphilic Foldamer[END_REF][START_REF] Rizkov | Donor-Acceptor-Promoted Gelation of Polyaromatic Compounds[END_REF] Evidence of copolymerization is primarily accomplished by demonstrating the inability of each monomer to homopolymerize. More recently, Besenius and co-workers demonstrated alternating supramolecular copolymers in aqueous solutions [START_REF] Frisch | pH-Switchable Ampholytic Supramolecular Copolymers[END_REF][START_REF] Ahlers | Dynamic Light Scattering Investigation of the Kinetics and Fidelity of Supramolecular Copolymerizations in Water[END_REF] and by sequential growth from Au-surface. [START_REF] Frisch | Kinetically Controlled Sequential Growth of Surface-Grafted Chiral Supramolecular Copolymers[END_REF][START_REF] Engel | Kinetic Control in the Temperature-Dependent Sequential Growth of Surface-Confined Supramolecular Copolymers[END_REF] Monomers based on benzene tricarboxamides bearing positively (1) and negatively (2) charged β-sheet encoded oligopeptides form alternating copolymers poly(1-alt-2) via electrostatic interactions (Figure 4a). Another example of peptide assembly has been reported by Giuseppone's group. [START_REF] Xiang | Hydrogen-Bonded Multifunctional Supramolecular Copolymers in Water[END_REF] Copolymerization in water between bisurea domains functionalized with bioactive peptide or fluorescent cyanine dyes resulted in fibers with an alternated fashion as established by fluorescence experiments. Working instead in the bulk, Araki and co-workers in 2003 reported the formation of alternating supramolecular copolymers between cyclohexane tricarboxamides functionalized with linear and bulky side chains. [START_REF] Takasawa | Steric-Factor-Directed Alternating Supramolecular Copolymer Composed of Hydrogen-Bonded Cyclohexanetricarboxamide Units[END_REF] The bulky monomer could not form stable homopolymers, but the authors found that it copolymerizes with the less bulky monomer in an alternating fashion based on infrared spectroscopy.

An interesting example reported by Li and co-workers [START_REF] Chen | Highly Stable Chiral (A)6-B Supramolecular Copolymers: A Multivalency-Based Self-Assembly Process[END_REF] relies on hierarchical organization to form an alternating supramolecular copolymer (Figure 4b). They employ zinc porphyrins that aggregate via complementary hydrogen bonding to form the hexamer (3)6. This assembly is geometrically compatible to react with the hexadentate monomer 4.

The authors use a combination of spectroscopic and static light scattering techniques to show that the monomers arrange in an alternating fashion to produce poly[(3)6-alt-4].

Periodic copolymers. Few examples of sequenced supramolecular copolymers have been reported. Würthner's group described in 2009 the formation of poly(A2-per-B) structured copolymers via unconventional interactions between complementary monomers: a perylene bisimide ( 6) and an azobenzene-functionalized melamine (5, Figure 5a). [START_REF] Yagai | Unconventional Hydrogen-Bond-Directed Hierarchical Co-Assembly between Perylene Bisimide and Azobenzene-Functionalized Melamine[END_REF] They reported that 5 and 6 do not form long organized structures in methylcyclohexane (MCH) in a 1:1 ratio as expected for ditopic 3-folded hydrogen bonding complementary units. Contrarily, the polymerization with a 2:1 ratio of 5:6 pectedly formed a periodic copolymer poly[(5)2-per-6] with J-type aggregation. The authors stressed the importance of the assembly protocol to acquire organized structures, as it required an equilibration time of 15 h at 20 °C. Atomic force microscopy (AFM) shows the presence of tightly coiled helical polymers with defined pitch (6 nm) and length (100-200 nm).

The Würthner group more recently reported how a related distorted perylene bisimide bearing imides protected with water-soluble chains ( 8) copolymerizes with a planar analogue (7) to form poly[(7)4-per-82] (Figure 5b). [START_REF] Görl | Supramolecular Block Copolymers by Kinetically Controlled Co-Self-Assembly of Planar and Core-Twisted Perylene Bisimides[END_REF] The homopolymerization of 7 in water follows an isodesmic mechanism to form stiff short nanorods via π-π interaction; in contrast, 8 forms dimers that aggregate into nanoworms via hydrophobic interactions. The copolymer is obtained under kinetic control by injecting water in a tetrahydrofuran (THF) solution of dissolved 8 and 7 in a 1:2 ratio, followed by the evaporation of the THF under ambient conditions. They assign the structure through a combination of optical microscopy, nuclear magnetic resonance (NMR) spectroscopy and transmission electron microscopy (TEM). Interestingly, poly[(7)4-per-( 8)2] forms long and thin flexible fibers that deviate from the morphology of the two homopolymers, evidently because the combination of the flexibility of the dimers of 8 and the rigidity of the 7-based blocks. This behavior is reminiscent of covalent elastomers where soft and hard blocks alternate to give new function. [START_REF] Hepburn | Polyurethane Elastomers[END_REF] Block copolymers. Stitching covalent polymers together to form diblock and multi-block copolymers gives rise to properties that are more than the sum of their parts. The key to their function is their ability to microphase separate into hierarchical nanostructures, which is a function of the compatibility and the volume fraction ratio of the blocks. The promise of higher order structural control has motivated supramolecular chemists to create analogous supramolecular block copolymers. Again, the non-covalent nature of the bonds and the inherent difficulty to characterize such structures render this goal a great challenge.

Seminal works have exploited kinetic control to retard exchange dynamics enough to furnish supramolecular block copolymers via sequential addition of monomers. In 2007, Winnik, Manners and co-workers reported the synthesis of supramolecular triblock copolymers of the form poly(A-b-Bb-A) using poly(ferrocenyldimethylsilane) (PFS) block copol-ymers as the supramolecular monomeric units. [START_REF] Wang | Cylindrical Block Copolymer Micelles and Co-Micelles of Controlled Length and Architecture[END_REF] By a kinetically controlled process that they named living crystallization, they were able to create short cylindrical homomicelles that exhibit uniform growth upon the addition of more monomer. They achieved the triblock architecture with control over the lengths of the blocks through the addition of a different dissolved PFS monomer. These aggregates are stable over time because of their low dynamicity. In contrast to the high dispersity observed for most supramolecular polymers produced through a cooperative mechanism, these structures exhibit low dispersity because of the "living" nature of this process, which was convincingly proven through TEM, static and dynamic light scattering (SLS, and DLS).

Research on PFS-based block copolymers has since flourished. Recent examples include the formation of regular crossed co-micelles, [START_REF] Qiu | Tunable Supermicelle Architectures from the Hierarchical Self-Assembly of Amphiphilic Cylindrical B-A-B Triblock Co-Micelles[END_REF] di-and tri-block co-micelles, [START_REF] Rupar | Non-Centrosymmetric Cylindrical Micelles by Unidirectional Growth[END_REF] fluorescent multi-block micelles, [START_REF] Hudson | Colour-Tunable Fluorescent Multiblock Micelles[END_REF][START_REF] Xu | Competitive Self-Assembly Kinetics as a Route to Control the Morphology of Core-Crystalline Cylindrical Micelles[END_REF] co-micelles between linear and brush units, [START_REF] Finnegan | Gradient Crystallization-Driven Self-Assembly: Cylindrical Micelles With "patchy" segmented Coronas via the Coassembly of Linear and Brush Block Copolymers[END_REF] photo-responsive systems, [START_REF] Gao | Crystallization-Driven Solution Self-Assembly of Block Copolymers with a Photocleavable Junction[END_REF] and multidimensional micelles. [START_REF] Li | Non-Covalent Synthesis of Supermicelles with Complex Architectures Using Spatially Confined Hydrogen-Bonding Interactions[END_REF][START_REF] Li | Cross" Supermicelles via the Hierarchical Assembly of Amphiphilic Cylindrical Triblock Comicelles[END_REF][START_REF] Hudson | Tailored Hierarchical Micelle Architectures Using Living Crystallization-Driven Self-Assembly in Two Dimensions[END_REF] Other types of monomer have been found to also undergo living crystallization, including poly(pphenylvinylene)s [START_REF] Shin | Living Light-Induced Crystallization-Driven Self-Assembly for Rapid Preparation of Semiconducting Nanofibers[END_REF] and poly(di-n-hexylfluorene)s. [START_REF] Jin | Long-Range Exciton Transport in Conjugated Polymer Nanofibers Prepared by Seeded Growth[END_REF] The latter, based on electron poor (9) and electron rich (10) poly(din-hexylfluorene)s, copolymerizes to form poly(10-b-9-b-10), which exhibits long range exciton transport (Figure 6a). Pavan, Takeuchi, Sugiyasu, and co-workers [START_REF] Sugiyasu | A Block Supramolecular Polymer and Its Kinetically Enhanced Stability[END_REF] recently demonstrated a seeded-growth copolymerization strategy to create kinetically stable supramolecular block copolymers. They generate seeds by sonicating homopolymers of zincbased porphyrin 11, and these seeds can be used to again grow poly(11) by adding additional 11 or to produce poly(12b-11-b-12) by adding the copper porphyrin 12 (Figure 6b). This process yields aggregates with a predictable length based on the monomer/seed ratio along with a narrow polydispersity. Remarkably, the triblock copolymer exhibits greater stability toward the presence of a denaturant compared to the poly(11) homopolymer. Molecular dynamics simulations reveal that monomer exchange only occurs at the end groups of the polymer, suggesting that the more stable poly(12) end blocks compartmentalize and protect the poly(11) mid-block.

Another exceptional example is presented by Aida's group, [START_REF] Zhang | Supramolecular Linear Heterojunction Composed of Graphite-like Semiconducting Nanotubular Segments[END_REF] who reported the block copolymerization of hexabenzacoronenes based-monomers through sequential steps (Figure 6c). The bipyridine-terminated hexabenzacoronenes units (13) homopolymerize in methanol to give bundles of poly(13). Metallation of the pendant bipyridine units with copper results in a dispersion of charged assemblies poly(13*). These aggregates are then dispersed in acetone and fragmented by sonication to create seeds. Upon adding 14 and cooling from 50 °C to 25 °C, the seeds initiate the supramolecular polymerization of 14 to give a mixture of diblock and triblock copolymers, poly(13*-b-14) and poly(14-b-13*-b-14), respectively.

Similarly to the synthesis of covalent block copolymers, the majority of supramolecular block copolymers are produced via a kinetic approach in which supramolecular polymers with low dynamicity can be fed with different monomers to form block structures. Their thermodynamic stability is in most cases limited, though. In contrast to these reports, we prepared supramolecular block copolymers under thermodynamic control with tripyridylamine tricarboxamide 15 and triphenylamine tricarboxamide 16. We found that supramolecular multiblock copolymers poly(15-co-16) are produced both by directly mixing the homopolymers poly (15) and poly( 16) and also by copolymerization of the molecularly dissolved monomers via a cooling procedure (Figure 6d). [START_REF] Adelizzi | Supramolecular Block Copolymers under Thermodynamic Control[END_REF] By combining optical spectroscopy and microscopy with the theoretical model developed for two-component supramolecular polymerization, we were able to identify the microstructure of the polymer, finding that the block length distribution is closely related to the enthalpy of co-interaction of the different monomers with respect to the enthalpy of the related homo-interaction.

Random and unresolved structures. Some of the earliest studies in the field of one-dimensional supramolecular polymerization consist of copolymerization experiments. Inspired by the work of Green and co-workers [START_REF] Green | Macromolecular Stereochemistry: A Cooperative Deuterium Isotope Effect Leading to a Large Optical Rotation[END_REF][START_REF] Green | Dilute-Solution Chiral Optical Changes Signal the Thermally Reversible Gelation of Poly(n-Hexyl Isocyanate) in Hydrocarbon Solvents[END_REF][START_REF] Green | A Helical Polymer with a Cooperative Response to Chiral Information[END_REF] on helical covalent polymers, these experiments were designed to probe the amplification of chirality in helical onedimensional supramolecular polymers. Typical experiments include mixing enantiomers of a monomer together (majority rules experiment), [START_REF] Van Gestel | Majority-Rules" operative in Chiral Columnar Stacks of C3-Symmetrical Molecules[END_REF][START_REF] Jin | Self-Assembled Graphitic Nanotubes with One-Handed Helical Arrays of a Chiral Amphiphilic Molecular Graphene[END_REF][START_REF] Langeveld-Voss | Principles of "Majority Rules" and "Sergeants and Soldiers" Applied to the Aggregation of Optically Active Polythiophenes: Evidence for a Multichain Phenomenon[END_REF][START_REF] Isare | Chirality in Dynamic Supramolecular Nanotubes Induced by a Chiral Solvent[END_REF][START_REF] Stals | Dynamic Supramolecular Polymers Based on Benzene-1,3,5-Tricarboxamides: The Influence of Amide Connectivity on Aggregate Stability and Amplification of Chirality[END_REF] mixing achiral monomers with chiral enantiopure analogues (sergeants and soldiers experiment), [START_REF] Langeveld-Voss | Principles of "Majority Rules" and "Sergeants and Soldiers" Applied to the Aggregation of Optically Active Polythiophenes: Evidence for a Multichain Phenomenon[END_REF][START_REF] Palmans | Sergeants-and-Soldiers Principle in Chiral Columnar Stacks of Disc-Shaped Molecules with C3 Symmetry[END_REF][START_REF] Kim | Dynamic Propeller Conformation for the Unprecedentedly High Degree of Chiral Amplification of Supramolecular Helices[END_REF][START_REF] Smulders | Insight into the Mechanisms of Cooperative Self-Assembly: The "Sergeants-and-Soldiers" Principle of Chiral and Achiral C3-Symmetrical Discotic Triamides[END_REF][START_REF] Van Gorp | C 3 -Symmetrical Supramolecular Architectures: Fibers and Organic Gels from Discotic Trisamides and Trisureas[END_REF][START_REF] Lohr | Time-Dependent Amplification of Helical Bias in Self-Assembled Dye Nanorods Directed by the Sergeants-and-Soldiers Principle[END_REF][START_REF] Ajayaghosh | Transcription and Amplification of Molecular Chirality to Oppositely Biased Supramolecular π Helices[END_REF][START_REF] Brunsveld | Hierarchical Formation of Helical Supramolecular Polymers via Stacking of Hydrogen-Bonded Pairs in Water[END_REF][100][101][102][103] and mixing chiral enantiopure monomers in different ratios with an achiral counterpart (diluted majority rules experiment). [104][105][106] The monomers in these cases typically comprise identical cores and only differ in the structure of their side chains. The mixtures were predominantly characterized using spectroscopic measurements such as fluorescence, ultraviolet-visible (UV-Vis) spectroscopy and especially circular dichroism (CD). Apart from some notable exceptions, 107 very few studies have corroborated the spectroscopic results with other types of characterization techniques. Usually, a stochastic incorporation of the monomers during the copolymerization process is assumed, but it has been difficult to experimentally assess if monomer incorporation is indeed fully random. Interestingly, several supramolecular copolymer systems exhibit a linear response in optical activity as a function of e.e. (majority rules) or percentage of chiral sergeant added (sergeant and soldiers). This lack of chiral amplification is a useful indicator for self-sorting (Figure 2), as it points to helical supramolecular homopolymers coexisting rather than mixing into copolymers. Detailed investigations by the groups of Würthner, Sanchez and our group suggest that these systems are often kinetically trapped, requiring either heat [START_REF] Lohr | Time-Dependent Amplification of Helical Bias in Self-Assembled Dye Nanorods Directed by the Sergeants-and-Soldiers Principle[END_REF]108,109 or the addition of a co-solvent 110 to initiate monomer exchange.

More recently, unresolved copolymer structures have been reported with monomers that have different supramolecular units [START_REF] Van Gorp | C 3 -Symmetrical Supramolecular Architectures: Fibers and Organic Gels from Discotic Trisamides and Trisureas[END_REF] or have different cores. [START_REF] Stals | Dynamic Supramolecular Polymers Based on Benzene-1,3,5-Tricarboxamides: The Influence of Amide Connectivity on Aggregate Stability and Amplification of Chirality[END_REF][START_REF] Van Gorp | C 3 -Symmetrical Supramolecular Architectures: Fibers and Organic Gels from Discotic Trisamides and Trisureas[END_REF]111 For example, the copolymerization of a chiral benzene tricarboxamide with an achiral benzene trisurea forms a kinetically trapped copolymer that spontaneously evolves into self-sorted homopolymers after 1 h. [START_REF] Van Gorp | C 3 -Symmetrical Supramolecular Architectures: Fibers and Organic Gels from Discotic Trisamides and Trisureas[END_REF] In contrast, the copolymerization of Ncentered and CO-centered benzene tricarboxamides most likely results in an alternating copolymer based on their donor-acceptor structure and chiral amplification studies. [START_REF] Stals | Dynamic Supramolecular Polymers Based on Benzene-1,3,5-Tricarboxamides: The Influence of Amide Connectivity on Aggregate Stability and Amplification of Chirality[END_REF] Finally, mixtures of copolymers of oligo(p-phenylvinylene) with different sizes and opposite chirality assemble into homopolymers with either preferred P-helicity or M-helicity at room temperature. Heating followed by slow cooling induces the formation of copolymers with the helical sense bias depending on the composition of the mixture. 111 In all of these cases, we rely on chemical intuition to propose their microstructures, but current analytical tools are insufficient to unambiguously assign their structures.

In some cases, the steric bulk of a monomer's side chains is too demanding, precluding it from mixing with another monomer to form a copolymer. 104,112 However, our group recently reported that a dendronized-pegylated benzene tricarboxamide is too bulky to homopolymerize but forms stable aggregates when mixed with a linearly pegylated benzene tricarboxamide in water. 113 A 2:1 ratio of dendronized:linear monomer exhibits a higher degree of order, slower dynamics, and higher stability compared to the homopolymer of the linearly pegylated monomer based on UV-Vis spectroscopy, hydrogen-deuterium exchange experiments, and cryoTEM imaging. Although an experimental elucidation of its structure remains elusive, molecular dynamic simulations suggest that a periodic 2:1 copolymer composition allows for an optimal balance between steric hindrance of the dendronized side chains and the stability afforded by intermolecular hydrogen bonds.

Another elegant system based on bipyridine-derived benzene tricarboxamides has been described by Brunsveld and co-workers as a functional material in biological systems. 114,115 Monomers with charged groups, mannose groups, fluorescent dyes, and biotin residues combine to form supramolecular copolymers in aqueous media, and the ratio of these components dictates their efficacy in binding and detecting bacteria. However, detailed information on the sequence and actual incorporation of the different monomers remains very challenging to obtain.

 CHARACTERIZATION CHALLENGES

The growing interest in developing non-covalent copolymers requires parallel progress and standardization of characterization techniques. As discussed above, such uniformity remains elusive. Other established fields, such as covalent copolymers, teach us that having a systematic, universally applied characterization routine is crucial to achieve concrete progress. However, the dynamicity of supramolecular polymers and the non-covalent nature of the bonds between the monomers render microstructural analyses extremely challenging. Nevertheless, nowadays a clear trend on the supramolecular homopolymers characterization routine has emerged. The prevalent strategy combines spectroscopic measurements (especially useful for variable temperature or variable solvent measurements), microscopy (the most used are AFM, TEM, cryoTEM, and super resolution fluorescence microcopy), mass spectrometry, and light scattering techniques. These experimental techniques are complemented with molecular dynamics simulations and thermodynamic mass-balance models.

The outstanding reports on supramolecular copolymers in which a detailed structure analysis is reported [START_REF] Adelizzi | Supramolecular Block Copolymers under Thermodynamic Control[END_REF][START_REF] Zhang | Supramolecular Linear Heterojunction Composed of Graphite-like Semiconducting Nanotubular Segments[END_REF]116 adapt and combine these techniques to resolve the supramolecular copolymer microstructure. Spectroscopy measurements (UV, CD, fluorescence, etc.) are powerful tools to unravel both the copolymerization mechanism [START_REF] Adelizzi | Supramolecular Block Copolymers under Thermodynamic Control[END_REF] and composition 117 of a given system by quantifying changes in spectroscopic features. Thereafter, spectroscopic results can be used in combination with theoretical modeling to predict the microstructure and the relative stability of the copolymer with respect to the homopolymers. [START_REF] Adelizzi | Supramolecular Block Copolymers under Thermodynamic Control[END_REF]117 The theoretical microstructural analysis can then be corroborated with judicious use of microscopy techniques. If applicable, TEM has proven to be an excellent tool to unravel structure. [START_REF] Zhang | Supramolecular Linear Heterojunction Composed of Graphite-like Semiconducting Nanotubular Segments[END_REF] Otherwise, recent developments in super resolution fluorescence microscopy demonstrate that it can be helpful in unravelling polymer growth 118 , dynamics [START_REF] Albertazzi | Probing Exchange Pathways in Super-Resolution Microscopy[END_REF]119 and structure. [120][121][122] Furthermore, hydrogendeuterium exchange followed by mass spectrometry is a valuable tool for following the dynamics of supramolecular systems in water. 123,113 What is striking from these seminal works is the lack of a leading experimental technique to unambiguously determine a copolymer's structure and the consequential need to bridge this gap with a combination of multiple techniques. This situation differs from that of covalent copolymers, where 1 H and [START_REF] Weng | Control of Gel Morphology and Properties of a Class of Metallo-Supramolecular Polymers by Good/Poor Solvent Environments[END_REF] C NMR spectroscopy are the primary tools for microstructural determination. We believe that further studies for developing techniques which allow similar analyses for supramolecular analogues are needed and will permit more efficient growth in the field. Microscopy, modeling, and molecular dynamics simulations have so far resulted in promising strategies. More progress in these directions could lead towards the possibility of analyzing in detail the subtle structural details of supramolecular copolymers.

 APPLICATIONS AND FUTURE PERSPECTIVES

The development of covalent copolymers has proven to be invaluable with time. Covalent copolymerization has opened the door to creating materials with properties simply not achievable with homopolymerization or blending. Considering supramolecular copolymers in this framework, we can dream of the advancements achievable through mastering dynamic multicomponent structures. Their properties are particularly well suited for managing complexity through adaptability. For biological applications, the prospect of including multiple units-such as sensors, bio-active molecules, and catalysts-in a defined order in non-covalent systems would be a breakthrough for synthetic bio-systems that require cooperative feedback between multiple systems. 124,125,114 The same effects can be exploited in organic electronics where tuning the arrangements of dynamic copolymers would optimize the optoelectronic properties of, for example, photoelectronic switches, sensors and chiral devices. [126][127][128] This perspective is meant to initiate a cooperative effort to advance this new and promising field. Despite the great progress achieved in the last years, much more must come. We believe that the field of supramolecular copolymerization needs comprehensive growth spanning strategic molecular design to the exploitation of theoretical models to the development of powerful characterization techniques. Achievable through collaboration and standardized analytical routines, these efforts will bring supramolecular copolymers to be essential in cutting edge technology markets.

Figure 1 :

 1 Figure 1: Growth mechanisms for chain polymerization. a) Covalent polymer activated by an initiator, which subsequently undergoes propagation or termination. b) Supramolecular polymer formed by a cooperative mechanism, which is activated by the unfavorable formation of a nucleus and elongated by the favorable incorporation of additional monomers.

Figure 2 .

 2 Figure 2. Covalent and supramolecular chain copolymers. General mechanism for a) covalent and b) supramolecular chain copolymerizations. c) Possible microstructures for each type of copolymerization. Polymers are ordered from no inter-reactivity (blend for covalent and self-sorted for supramolecular polymers) to high inter-reactivity (alternate copolymers).

Figure 3 .

 3 Figure 3. Supramolecular copolymerization model. a) Terminal model of the supramolecular copolymerization between two types of monomers. The model assumes that the equilibrium constant depends only on the identity of the terminal monomer unit (PnA or PnB). b) The mass balance defines the concentration of polymerized monomer [M]pol as the difference between the total concentration of monomer [M]tot and the free monomer [M]free. The fraction of polymerized monomer is defined as φpol. c) The composition and microstructure of the copolymer is determined by the reactivity ratio R, which is defined by the ratio of Gibbs free energy change of the homo-aggregation (ΔGA-A + ΔGB-B) to the Gibbs free energy change of the hetero-aggregation (ΔGA-B + ΔGB-A. d) Copolymerization curves are reported as a function of R: from left to the right, descending order from R approaching infinite values for self-sorted systems to R smaller than 1. e) Equation defining the fraction of A in the polymer (FA). f) Plot of FA vs. temperature as a function of R.

Figure 4 .

 4 Figure 4. Examples of alternating supramolecular copolymers achieved with different strategies. a) Poly(1-alt-2) is primarily ordered by Coulombic interactions. b) The sequence of poly[(3)6-alt-4) is controlled by complementary hierarchical interactions.

Figure 5 .

 5 Figure 5. Examples of periodic supramolecular copolymers achieved with different strategies. a) Stable helical copolymers are obtained by unconventional hydrogen bonding interaction between complementary units of melaminefunctionalized azobenzene 5 and perylene bisimide 6 resulting in poly[(5)2-per-6]. b) Planar 7 and twisted 8 perylene bisimides copolymerize under kinetic control to form long copolymers poly[(7)4-per-(8)].

Figure 6 .

 6 Figure 6. Examples of supramolecular block copolymers. a) Covalent block copolymers 9 and 10 feature a crystalline midblock of poly(di-n-hexylfluorene) and are used as monomers to obtain triblock micelles poly(9-b-10-b-9) via seeded multistep addition and crystallization driven copolymerization. b) Supramolecular copolymerization of zinc porphyrin 11 followed by copper porphyrin 12 under kinetic control yields a triblock copolymer poly(11-b-12-b-11). c) Supramolecular block copolymers based on electron rich and electron poor hexabenzocorone-base monomers 13 and 14, respectively, are obtained by a multistep synthesis. c) Supramolecular multiblock copolymers are obtained between tripyridylamine tricarboxamide 15 and triphenylamine tricarboxamide 16 under thermodynamic control. Direct mixing of poly(16) to poly(15) or copolymerization from a mixture of monomers lead to the same copolymer microstructure poly[(15)-co-(14)].

 ACKNOWLEDGEMENTS

The authors like to thank Dr. H.M.M. ten Eikelder and Dr. A.J. Markvoort for fruitful discussions and the mathematical models describing the supramolecular copolymerizations. We acknowledge funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 705701 (N.J.V.Z.), the Dutch Science Foundation (NWO ECHO Grant No. 712.016.003, L.N.J.d.W.), and the Dutch Ministry of Education, Culture and Science (Gravity program 024.001.035).


Guillaume Lefebvre

Antoine Maze

Rosa Alvarez

Palencia Jimenez

Franz Bruckert

Vasco Filipe

Sylvain Huille

Marianne Weidenhaupt
email: marianne.weidenhaupt@grenoble-inp.fr

Rosa Alvarez-Palencia Jimenez

Marianne Weidenhaupt Phelma

Minatec Lmgp

Surfactant Protection Efficacy at Surfaces Varies with the Nature of Hydrophobic Materials

Keywords: monoclonal antibody, surface adsorption, formulation optimization, surfactant protection, plastic materials

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Monoclonal antibodies (mAbs) are complex proteins that have become increasingly important due to their numerous therapeutic applications, in autoimmune diseases or cancers [START_REF] Lu | Development of therapeutic antibodies for the treatment of diseases[END_REF]. The recent development of new mAb formats, such as bispecific antibodies and conjugated antibodies, further increases the interest of medical research for this kind of drug [START_REF] Kaplon | Antibodies to watch in 2020[END_REF].

During production, transport in primary packaging, like vials or prefilled syringes, or administration with intravenous (IV) infusion bags, monoclonal antibodies may adsorb at the liquid-solid interface and form aggregates in solution that can bring safety concerns [START_REF] Moussa | Immunogenicity of Therapeutic Protein Aggregates[END_REF].

However, while aggregate formation can be mitigated and easily monitored during mAb production, it is more difficult to predict the behaviour of therapeutic proteins during the transport, dilution and administration stages. In order to avoid any mAb surface adsorption during these steps, it is common to add surfactants to the drug formulation. Surfactants have the ability to protect therapeutic proteins by competitive adsorption at interfaces [START_REF] Khan | Key interactions of surfactants in therapeutic protein formulations: A review[END_REF].

Polysorbate 80 (PS80), Polysorbate 20 (PS20) and Poloxamer 188 (P188) to a lower extent, are widely used in almost 90% of all marketed mAb formulations [START_REF] Gervasi | Parenteral protein formulations: An overview of approved products within the European Union[END_REF].

It has been shown that mAbs can adsorb to materials such as plastics, which are used for the manufacture of vials, syringe barrels, IV bags or tubing [START_REF] Zhang | Adsorption of polysorbate 20 and proteins on hydrophobic polystyrene surfaces studied by neutron reflectometry[END_REF][START_REF] Browne | Protein adsorption onto polystyrene surfaces studied by XPS and AFM[END_REF]. The sensing techniques used in interface adsorption studies are often not directly compatible with the plastic materials in contact with therapeutic solutions. Instead these studies are conducted on wellcontrolled hydrophobic surface functionalizations, mimicking the overall hydrophobic character of plastics [START_REF] Kapp | Competitive adsorption of monoclonal antibodies and nonionic surfactants at solid hydrophobic surfaces[END_REF][START_REF] Couston | Adsorption behavior of a human monoclonal antibody at hydrophilic and hydrophobic surfaces[END_REF]. However, plastic materials are not defined exclusively by their hydrophobicity. They are obtained by more or less ordered polymerisation of repeated monomer units using processes involving heat, pressure and the addition of catalysts and plasticizer. Their nature and fabrication processes allow tuning different functionalities, such as flexibility, moisture permeation, transparency, collapsibility and compatibility with heat sterilisation, which are relevant in medical devices [START_REF] Sacha | Practical fundamentals of glass, rubber, and plastic sterile packaging systems[END_REF]. The most used ones in medical applications are polystyrene, polyolefins (polypropylene, polyethylene), polyvinylchloride and polycarbonate [START_REF] Basle | Physicochemical Stability of Monoclonal Antibodies: A Review[END_REF].

The influence of the properties of different plastics on the adsorption potential of mAbs at their surface is hitherto poorly understood. We therefore compared the adsorption and desorption kinetics of three monoclonal antibodies in the presence of PS80, PS20 and P188 on hydrophobic model surfaces (hexa and octadecane-based) and polystyrene, using surface-sensitive techniques, such as surface plasmon resonance imaging (SPRi) and quartz crystal microbalance with dissipation monitoring (QCM-d). We show that the protective effect of surfactants differs significantly on the model surfaces and on polystyrene, despite their similar hydrophobicity. Additionally, we used an enzyme-linked immunosorbent assay to study the protective effect of surfactants on a number of different plastics and show that it varies significantly with the nature of the plastic surface and depends on the mAbsurfactant ratio.

Experimental Proteins and Chemicals

Monoclonal antibodies A, B and C

Monoclonal antibody A (mAbA) is a full IgG of subclass 1, provided by Sanofi (Paris, France) at 30 g.L -1 in a formulation at pH 6.0 containing histidine and sucrose; mAbA is positively charged at pH 6.0. mAbB is a bispecific antibody fragment construct derived from a subclass 1 IgG provided by Sanofi (Paris, France) at 0.7 g.L -1 in a formulation at pH 6.0 containing histidine, glycine and sucrose; mAbB is positively charged at pH 6.0. mAbC is a full IgG of subclass 4, provided by Sanofi (Paris, France) at 3 g.L -1 in a formulation at pH 6.1 containing histidine, sucrose and proline; mAbC is also positively charged at pH 6.1.

The charge and hydrophobicity characteristics of the three mAbs are published [START_REF] Lefebvre | Adsorption rate constants of therapeutic proteins and surfactants for material surfaces[END_REF].

The buffers of the respective mAbs are hereafter named Bf 1x. Concentrated mAb aliquots were stored at -20°C and thawed shortly before use. When thawed, mAb solutions were kept at 4°C. They were diluted in their respective Bf 1x to the working concentrations. The

Dynamic light scattering

The absence of protein aggregates was verified for mAb solutions by dynamic light scattering (DLS) on a Malvern Zetasizer Nano instrument. mAbA was measured at 30 g.L -1 , mAbB at 0.7 g.L -1 and mAbC at 3 g.L -1 . The data were acquired using the ZSExplorer size protocol (30 acquisitions, total time 50.4s) and analysed with ZSExplorer software (general purpose model, adaptive correlation mode). From the size distribution by intensity, the hydrodynamic diameter was deduced based on the Stokes-Einstein equation and was 10 nm as expected for mAbA, mAbB and mAbC with low polydispersity index values (< 0.13).

Surface functionalizations

The sensor surface of gold-coated prisms (Horiba SPRi Biochips) was functionalized according to a protocol described by Nault [START_REF] Nault | Human insulin adsorption kinetics, conformational changes and amyloidal aggregate formation on hydrophobic surfaces[END_REF] to obtain a hydrophobic surface, coated with hexadecane thiol (C16-SH, Sigma-Aldrich), diluted to 1.6% in toluene and with a thiolterminated polystyrene (P10826A-SSH, Polymer source), diluted to 1% in toluene.

In the same way, gold-coated QCM-d sensors (QSX301) from Biolin Scientific (Västra Frölunda, Sweden) were functionalized to obtain a hydrophobic surface, coated with hexadecane thiol (C16-SH, Sigma-Aldrich), diluted to 1.6% in toluene. The polystyrene QCMd sensors (QSX305) were purchased from Biolin Scientific.

Borosilicate glass beads (1mm -Z273619, Sigma-Aldrich) were cleaned by rinsing with 14.5M NaOH followed by repeated rinsing with deionized water and drying at 100°C for 1h. Cleaned glass beads were functionalized by immersion during 2 hours with gentle agitation in octadecyl methyl dichlorosilane (C18) (AB111265, ABCR), diluted to 0.1% in toluene. Then, the functionalized beads were washed with toluene, ethanol and water and cured at 100°C for 1 hour.

Surface characterisation

X-Ray Photoelectron spectra were recorded on a ThermoScientific K-Alpha spectrometer using a monochromatized Al K radiation source (1486.6 eV), in ultra high vacuum (10 -8 mbar) at room temperature. The X-Ray beam area (spot size) was adjusted to 400 μm in diameter. Analyses were conducted at 90° angles between the sample surface and the analyzer. The photoelectrons emitted were inspected with a 180° double focusing hemispherical analyzer and a 128-channel detector. The spectra were acquired in the constant analyzer energy mode using a pass energy of 30 eV and a step size of 0.1 eV for core levels, and a 100 eV pass energy has been used for the general survey spectrum (step 0.5 eV). The spectra shown in the figures have been averaged over 3 scans for the survey and 10 scans for the core levels. The flood gun was used to neutralize charge effects on the surface. All the binding energies were calibrated relatively to the C-C bond component of the C 1s core electrons at 284.6eV from adventitious carbon adsorbed on the sample surface.

The inelastically scattered electron-related background signal has been substracted by the Shirley method. Peak analysis and decompositions were performed using Thermo Scientific Avantage software.

Plastic multiwell plates

Polystyrene (655101) and polypropylene (650201) 96-well microplates were purchased from Greiner and polycarbonate (6520), polyvinylchloride (2595), cyclic olefin copolymer (4680) and PEG-coated (3651) 96-well microplates were purchased from Corning. The water contact angles (WCA) on these plastics were measured with a drop shape analyzer (DSA100 Krüss Scientific) and were as follows: Polystyrene WCA = 88.9 ± 3.2; polypropylene WCA = 90.0 ± 2.0; polycarbonate WCA = 82.2 ± 4.9; polyvinylchloride WCA = 95.9 ± 3.4; cyclic olefin copolymer WCA = 85.9 ± 1.0.

Surface plasmon resonance imaging (SPRi)

Sensogram acquisition

Reflectivity data were obtained with an SPRi instrument (Horiba Scientific SPRi-Lab+™) using an established protocol [START_REF] Lefebvre | Adsorption rate constants of therapeutic proteins and surfactants for material surfaces[END_REF]. Buffer, protein and surfactant solutions were prepared at room temperature and degassed under vacuum before use.

The amount of stably surface-adsorbed molecules was determined after rinsing with Bf1x and stabilization of the signal by measuring the resonance signal relative to the baseline and is indicated as ΔR. SPRi signals, recorded every 3 seconds from each spot, were used to calculate a mean value and standard deviation for each measurement. For each experimental condition, three independent experiments were performed.

Quartz crystal balance with dissipation monitoring (QCM-d)

Protein and surfactant adsorption were measured on a Q-Sense E4 system QCM-d instrument (Biolin Scientific, Västra Frölunda, Sweden). Buffer, protein and surfactant solutions were prepared at room temperature and degassed under vacuum before they were injected in the QCM-d chambers using a peristaltic pump.

Before each experiment, the crystal surface was cleaned successively by sonication in 2% SDS and absolute ethanol for 10 minutes each. The baseline was set by the frequency obtained with Bf1x, corresponding to each protein. After baseline stabilization, protein and surfactant solutions were injected at the indicated concentrations in their respective Bf 1x.

The flow rate (30 L.min -1 ) was kept constant during the experiment. Protein and surfactant solutions were injected until the frequency signal reached a steady state. The amount of stably surface-adsorbed molecules was determined after rinsing with Bf1x and stabilization of the signal by measuring the frequency signal relative to the baseline and is indicated as ΔF. For each experimental condition, two independent experiments were performed. and surfactants, we analyzed the adsorption of 3 different mAbs on the same surface chemistries using an ELISA assay. The adsorption of mAbA at 30g.L -1 , mAbB at 0.7g.L -1 and mAbC at 3g.L -1 (concentrations of formulated mAbs), was analysed as a function of PS80 concentration in a PS multi-well plate and on octadecane (C18)-functionalized glass beads in a protein-repellent polyethylene glycol (PEG)-coated multiwell plate. Note that in these experiments the surface area in contact with the solution limits the amount of protein adsorption, resulting in similar adsorption levels despite significantly different mAb bulk concentrations. On PS, the protective effect of PS80 for all mAbs tested increases with the concentration of the surfactant and is strongest at 0.2g.L -1 , when mAbA, mAbB and mAbC remain adsorbed at 2%, 7.5% and 6%, respectively, relative to the condition without surfactant (Figure 3a). On C18-functionalized glass beads, however, the protective effect is minimal and mAbA, mAbB and mAbC remain adsorbed at 71%, 75% and 88%, respectively, at 0.2g.L -1 of surfactant (Figure 3a), relative to their adsorption level in the absence of surfactant. The amount of each mAb, adsorbing directly to PEG-coated wells, was shown to be low, relative to the amount measured on C18-functionalized beads in PEG-coated wells (17% for mAbA, 12 % for mAbB and 13 % for mAbC, Supplementary Figure 3a). Despite different overall hydrophobicities for the three mAbs [START_REF] Lefebvre | Adsorption rate constants of therapeutic proteins and surfactants for material surfaces[END_REF], these results confirm that the protective effect of PS80 is strongly dependent on the material surface.

In Figure 3b, we extend the results obtained with PS80 to PS20 and P188. On C18functionalized beads, the mAbA adsorption in the presence of 0.2g.L -1 surfactant is 71%, 61% and 53% for PS80, PS20 and Poloxamer 188, respectively, compared to the condition without surfactant. On PS plates, mAbA remains adsorbed at 5%, 3% and 9% in the presence of 0.2g.L -1 of PS80, PS20 and P188, respectively, relative to the adsorption level without surfactant. We therefore conclude that the protective effect of surfactants differs depending on the material surface, and this is true for all antibodies and surfactants tested.

The protective effect of PS80 varies on different plastic materials

We further broadened our findings to other, commercially available, plastics, notably polypropylene (PP), polyvinylchloride (PVC), cyclic olefin copolymer (COC) and polycarbonate (PC) using the ELISA-based assay (Figure 4). The absorbance data (Figure 4a) were obtained by a colorimetric readout using PtG/PtA-HRP which specifically binds to surface-adsorbed mAbA. Absorbance levels for mAbA without surfactant are in a similar range for the different materials, except for the PC plate, on which they are significantly lower. This is due to the fact that the surface area in contact with the solution in PC plates is 50% smaller and the well volume is reduced to 50 L in these plates. In formulation conditions (30g.L -1 mAbA and 0.2 g.L -1 PS80), the surfactant shows very good protection on PS and PP and PC with 1.7, 4.4 and 3.6% mAbA adsorption, respectively, compared to the adsorption level without surfactant (Figure 4a andb). On PVC, the amount of adsorbed mAbA is reduced to 48.4 %, illustrating an intermediate protection effect of PS80 (Figure 4a andb). mAbA adsorption could not be prevented by PS80 on COC plates in formulation conditions. Very similar results were also obtained with PS80 from Croda (Supplementary Figure 4), illustrating that, within pharmacopeia limits, the variations in the chemical compositions of surfactants from different suppliers do not change their protective efficacy on the tested plastics.

Interestingly, the PtG-HRP construct showed a high level of non-specific binding to the COC surface in the absence of mAbA. ELISA assays on COC plates were therefore performed with a PtA-HRP construct. Both PtG-and PtA-HRP constructs showed equal performances in the ELISA assays on PS plates (Supplementary Figure 3b), indicating that mAb surface adsorption is unlikely to affect binding site accessibility for PtG.

Discussion

The control of molecular surface interactions is pivotal in therapeutic formulation development as well as in compatibility assessment for materials in contact with blood cells in vitro and in the human body. This is particularly the case for a plethora of hydrophobic plastic materials in contact with therapeutic formulations from their production, all the way to their administration [START_REF] Basle | Physicochemical Stability of Monoclonal Antibodies: A Review[END_REF]. In-use plastic materials are often mimicked in experimental studies by hydrophobic model surfaces, which have the advantage to be easily implemented with interface-sensitive techniques [START_REF] Prime | Self-assembled organic monolayers: model systems for studying adsorption of proteins at surfaces[END_REF], but are not fully representative of the diversity of the chemical and physical properties of plastics. Our study compares directly mAb and surfactant adsorption profiles on a self-assembled alkyl model monolayer (C16) and on polystyrene (PS) by SPRi and QCM-d. We further extend our investigations on surface adsorption and surfactant protection to commercially available plastics using an adapted ELISA protocol.

PS80 adsorbs as expected irreversibly on the hydrophobic surfaces, slightly more on the PS surface compared to the C16 surface (Figure 1 and Table I). This corroborates the findings of Zhang and colleagues, who have demonstrated, by QCM-d and neutron reflectometry, that another polysorbate, PS20, adsorbs fast and irreversibly on a PS surface [START_REF] Zhang | Adsorption of polysorbate 20 and proteins on hydrophobic polystyrene surfaces studied by neutron reflectometry[END_REF]. PS20 also irreversibly adsorbs on silicone oil, a hydrophobic lubricant often used in contact with therapeutic solutions [START_REF] Dixit | The effect of Tween® 20 on silicone oil-fusion protein interactions[END_REF]. Subsequent injection of mAbA at 30 g.L -1 results in the accumulation of a significantly higher mass on C16, precoated with PS80, than on PS, precoated in the same way (Figure 1 and Table I). Using PtG, which selectively binds to immunoglobulins and shows very low nonspecific binding to both C16 and PS surfaces precoated with PS80 (Figure 2), we demonstrate that, in formulation conditions, PS80 cannot prevent irreversible mAb binding on both C16 and PS, albeit in different proportions.

The protective effect of PS80 is significantly better on PS with mAbA adsorption reduced to 46% (SPRi) and 32% (QCM-d), relative to mAbA adsorption without surfactant (100%). The amount of mAbA adsorbing on a C16 model surface in the same formulation conditions, is 98% (SPRi) and 90 % (QCM-d) compared to mAbA adsorption without PS80 (100%) (Figure2).

Commonly, adsorption studies compare hydrophobic and hydrophilic model surfaces when analyzing the protective effect of surfactants on protein adsorption [START_REF] Joshi | Adsorption behavior of lysozyme and tween 80 at hydrophilic and hydrophobic silica-water interfaces[END_REF]. The comparison with protective efficacy of surfactants on hydrophobic surfaces present in the literature is difficult, because individual studies present data with different molecular systems, experimental protocols and techniques. Our results differ from the conclusions of Kapp and colleagues [START_REF] Kapp | Competitive adsorption of monoclonal antibodies and nonionic surfactants at solid hydrophobic surfaces[END_REF], who demonstrated by QCM-d and total internal reflection fluorescence microscopy (TIRF), that pre-adsorbed PS80 on a trifluoropropyl-chloromethylsilane surface, (WCA = 89°), was able to prevent the adsorption of an IgG1 concentrated at 10g.L -1 . On the other hand, Couston and colleagues demonstrated by TIRF, on an octadecyl-trichlorosilane surface (WCA = 107.1°), that a precoating with PS80 and PS20 did not completely prevent the adsorption of an IgG1 at significantly lower concentrations of 0.01 g.L -1 [START_REF] Couston | Adsorption behavior of a human monoclonal antibody at hydrophilic and hydrophobic surfaces[END_REF]. At the silicone oil-water interface, a pre-adsorption of PS20 is able to reduce protein adsorption significantly [START_REF] Dixit | The effect of Tween® 20 on silicone oil-fusion protein interactions[END_REF]. These differences reveal that, the protective efficacies of surfactants in preventing IgG1 surface adsorption strongly depend on complex material surface properties that cannot be described by a hydrophobic WCA measurement alone. Indeed, the hydrophobic materials considered in this study have WCAs ranging from 82° (PC) to 103° (C16), but there is no obvious correlation between the protective efficacy of the surfactant and the water contact angle of the materials (Supplementary Figure 5). The strength with which the surfactant (or protein) binds to a particular surface is likely to depend on a combination of properties like the nature and density of surface functional groups, the degree of crystallinity, the surface rugosity, and others. An indirect illustration of this is also given by the work of Browne and colleagues who demonstrated that on polystyrene, albumin adsorption decreased with an increasing oxidation state of the polymer surface [START_REF] Browne | Protein adsorption onto polystyrene surfaces studied by XPS and AFM[END_REF].

In order to compare the potential of surfactant protection for mAbs on a variety of plastic materials, we developed an ELISA assay, that is versatile and rapid and can be implemented directly on the chosen plastic. It differs from the surfactant pre-coating protocols in that the surfactant and antibody are applied simultaneously to the surface, mimicking formulation conditions. Using this approach, we extended our adsorption studies to two other mAbs (mAbB and mAbC, Figure 3a) and to PS20 and P188 (Figure 3b), two other surfactants often used in therapeutic formulations. Varying PS80 concentrations (0 to 0.2g.L -1 ) we show that, for each mAb at its respective formulation concentration, a significant difference in the amount of mAb surface adsorption is observed in a PS multi-well plate compared to octadecane (C18)-functionalized glass beads in a PEG-coated hydrophilic multi-well plate (Figure 3a). While the protection efficacy improves rapidly on PS with increasing PS80 concentrations for all antibodies tested (maximal protection at 0.2g.L -1 PS80), the protection efficacy of PS80 on C18 is minimal and does not improve significantly in the concentration range tested. This behaviour shows that on PS, contrary to C18, PS80 competes efficiently and dominates surface adsorption above a critical concentration. This has also been described for the protection efficacy of PS80 and lysozyme (0.5 g.L -1 ) adsorption on hydrophobic silica functionalized with dichlorodimethylsilane (WCA = 90-110°) for which maximal protection was measured at 0.08g.L -1 (80ppm) [START_REF] Joshi | Adsorption behavior of lysozyme and Tween 80 at hydrophilic and hydrophobic silica-water interfaces[END_REF]. When considering PS20 and more particularly P188 at 0.2g.L -1 , we show that their protection efficacies against mAbA surface adsorption is not significantly different than the one described for PS80 on both PS and C18 surfaces. In a study carried out on hydrophilic silica-titania surfaces, Kim and colleagues concluded that P188 inhibits protein adsorption by forming protein-surfactant complexes rather than by preferential surface adsorption which is the case for polysorbates [START_REF] Kim | Modulation of Protein Adsorption by Poloxamer 188 in Relation to Polysorbates 80 and 20 at Solid Surfaces[END_REF]. It was also shown that PS20 was more effective than P188 to prevent mAb adsorption at the silicone oil-water interface [START_REF] Kannan | Adsorption and Aggregation of Monoclonal Antibodies at Silicone Oil-Water Interfaces[END_REF]. Our results, based on the relative quantification of adsorbed mAbA on hydrophobic surfaces, do not suggest a different underlying protection mechanism for polysorbates and P188 and rather point towards equally efficient/inefficient surface interactions on PS/C18 for all surfactants tested.

We then quantitatively compared the protective effect of PS80 on different, commercially available plastic multi-well plates (Figure 4). The variability of the protection efficacy observed for the different plastic materials tested illustrates that a complex interplay of material properties dictates molecular surface binding for both proteins and surfactants and can thus result in strong or weak surfactant protection efficacies. Adsorption studies with "real materials" in use during the life cycle of a therapeutic molecule are quite rare because they are often not easily amenable to surface-sensing techniques. Zangmeister demonstrated by X-Ray photo-electron spectroscopy (XPS) that a mAb and a polyclonal human IgG could adsorb on a glass vial, a vial rubber cap liner, a syringe plunger tip, and a cell culture flask but not on PVC tubing nor on a plastic barrel of a syringe [START_REF] Zangmeister | Application of x-ray photoelectron spectroscopic analysis to protein adsorption on materials relevant to biomanufacturing[END_REF]. Different polymeric filter materials were tested for mAb and PS80 adsorption by Mahler and colleagues [START_REF] Mahler | Adsorption Behavior of a Surfactant and a Monoclonal Antibody to Sterilizing-Grade Filters[END_REF], who demonstrated that the amount of protein adsorption varies with filter material but also with different suppliers for the same material. In an attempt to control cell adhesion, the influence of surface functional groups in culture dish inserts made from various plastic polymers (PS, PC, polystryrene-co-acrylonitrile and polyetherimide) on IgG1 adsorption was analysed [START_REF] Bhaskar | Single and competitive protein adsorption on polymeric surfaces[END_REF]. Despite similar wettability and surface roughness, surfaceadsorbed protein amounts varied, with PS showing minimal adsorption and PEI showing the highest protein adsorption. The authors conclude that the chemical composition of the polymers allows for specific combinations of potential interaction forces, influencing protein adsorption profiles. Zheng and colleagues drew similar conclusions by reviewing the influence of surface charge, chemical composition, surface topography and crystalline phases in hydrophilic bioactive glasses on BSA adsorption [START_REF] Zheng | Protein interactions with bioactive glass surfaces: A review[END_REF]. Interistingly, Thyparambil and colleagues also showed that surfactant performance in eluting adsorbed proteins from surfaces not only depends on the detergent used but also on the type of surface [START_REF] Thyparambil | Evaluation of the Effectiveness of Surfactants and Denaturants to Elute and Denature Adsorbed Protein on Different Surface Chemistries[END_REF]. The consequences of differential surface adsorption in response to underlying material properties is also illustrated in different modes of surface-induced amyloid fibrillation [START_REF] Grigolato | The role of surfaces on amyloid formation[END_REF].

Conclusion

Our study confirms that inherent material properties of plastic polymers influence protein but also surfactant adsorption in such a way that it can greatly affect the protective efficacy of commonly used surfactants such as polysorbates or poloxamer 188. It is therefore crucial to include the evaluation of protection efficacies at targeted material interfaces in the formulation optimisation process. If materials can be provided in pieces with fitting dimensions compatible with analysis in multiwell plates, our ELISA-based assay represents a convenient screening protocol in formulation optimization for different materials relevant to therapeutic protein handling and administration. a, Adsorption recorded by SPRi during a sequential injection of PS80 at 0.2 g.L-1 and mAbA at 30 g.L-1 on a prism functionalized with polystyrene (grey) or hexadecane (black). ΔR PS80 and ΔR mAbA (double headed arrows) indicate the amount of PS80 and mAbA that remain adsorbed on the surfaces after a buffer wash, expressed in reflectivity units, RU (see Table 1). b, Adsorption recorded by QCM-d during a sequential injection of PS 80 at 0.2 g.L-1 and mAbA at 30 g.L-1 on a sensor functionalized with polystyrene (grey) or hexadecane (black). ΔF PS80 and ΔF mAbA (double headed arrows) indicate the amount of PS80 and mAbA that remain adsorbed on the surfaces after a buffer wash, expressed in Hertz, Hz (see Table I). polystyrene-coated prism. The PtG is injected after: a, an adsorption of PS80 at 0.2g.L -1 (R 0 ) followed by a buffer (B) wash, b, an adsorption of mAbA at 30g.L -1 without surfactant (R 1 ) followed by a buffer (B) wash and c, an adsorption of mAbA at 30g.L -1 with PS80 at 0.2g.L -1 (formulation condition (F)) (R 2 ) followed by a buffer (B) wash. d, Plot showing the amount of adsorbed PtG in formulation conditions normalized to the condition without surfactant on C16 (black) and PS (grey) by SPRi (left) and QCM-d (right). The results are expressed according to the formula (R 2 -R 0 ) /R 1 for the SPRi data and likewhise to the formula (F 2 -F 0 ) /F 1 for the QCM-d data (Supplementary Figure 1 and Supplementary Table I a, Absorbance at 450 nm for mAbA (30 g.L -1 ) adsorption without and with PS80 (0.2 g.L -1 ) and for PS80 (0.2 g.L -1 ) alone, in a polystyrene plate (PS, black), a polypropylene plate (PP, dark grey), a polyvinyl chloride plate (PVC, light grey), a cyclic olefin copolymer plate (COC, dotted) and a polycarbonate plate (PC, white). For the COC plate, a PtA-HRP construct was

  addition of different surfactants to the formulations was made by spiking a concentrated solution of the surfactant to reach the desired final concentration. The concentrations of mAb aliquots were verified by absorbance at 280 nm using a Nanodrop spectrophotometer. Protein G/A-HRP conjugates Protein G-HRP (Merck Ref 18-161) and protein A-HRP (GE Healthcare Ref NA9120V) conjugates were rehydrated at 1g.L -1 in MilliQ water. Concentrated protein aliquots were stored at -20°C and thawed shortly before use. When thawed, protein G and A conjugates were diluted at 250µg.L -1 in the mAb-specific buffers. Surfactants Polysorbate 80 (PS80) (Seppic Montanox 80ppi, MW = 1310 g.mol -1 ) and Polysorbate 20 (PS20) (Seppic Montanox 20ppi, MW = 1227 g.mol -1 ) are compliant with EP, USP and JP monographs. Polysorbates and Poloxamer 188 (P188) (Merck) were diluted at the indicated concentrations in the respective buffers (Bf1x), described above.

FiguresFig 1

 1 Figures

  Fig 1 Adsorption of PS80 and mAbA on polystyrene (PS) and on a hexadecane (C16)

Fig 2

 2 Fig 2 Effect of PS80 on the adsorption of mAbA to polystyrene or a hexadecane-

Fig 3

 3 Fig 3 Comparison of the protective effect of different surfactants and mAbs on polystyrene

  mAb adsorption is quantified by ELISA using a PtG-HRP construct. Data are obtained by absorbance measurements using a colorimetric enzymatic readout. They are normalized to the absorbance measured for mAbA adsorption without surfactant. Each data point corresponds to the mean of eight experiments and the error bars indicate the standard deviation.

Fig 4 PS80

 4 Fig 4 PS80 protective effect on different material surfaces

  diagram illustrating the protective effect of PS80 on the different plastic surfaces. Each bar represents the % of mAbA adsorbed in the presence of 0.2g.L -1 PS80, normalized to the condition without surfactant. The absorbance data of panel a were used for this 0 data point corresponds to the mean of eight experiments and the error bars indicate the standard deviation.

5 a

 5 Surface a F PS80 (Hz) c F mAbA -F PS80 (Hz) Adsorption of PS80 and mAbA on sensor surfaces functionalized with polystyrene and hexadecane as measured by SPRi and QCM-d (see Figure1). b Each SPRi value corresponds to the mean of three experiments and their standard deviation. c Each QCM-d value corresponds to the mean of two experiments and their standard deviation.

Table Table I Reflectivity and frequency data for adsorbed mAb and surfactant on different

 Table

Acknowledgements

The authors thank Matthieu Weber for excellent technical expertise with XPS acquisition.

Funding Statement

GL was supported by an ANRT Cifre PhD grant (2017/0735). This work was supported by the French National Research Agency in the framework of the "Investissements d'avenir" program (ANR-15-IDEX-02).

Enzyme Linked Immunosorbent Assay (ELISA)

The concept of the ELISA-based protocol relies on quantifying relative amounts of mAb that adsorb directly on different material surfaces. An enzymatically driven, colorimetric readout is obtained thanks to the specific binding of PtG/PtA-HRP. Absorbance is therefore directly proportional to the amount of surface-adsorbed mAb. We analyze absorbance values obtained for mAb surface adsorption without and with increasing amounts of surfactant.

ELISA experiments were performed directly in plastic multiwell plates (PS, PP, PVC, COC and PC) and on C18-functionalized beads in PEG-coated multiwell plates, which are protein repellent. The low level of protein binding in PEG-coated multiwell plates is documented in Supplementary Figure 3a. In each PEG-well, 31 C18-coated glass beads were added. The surface area of 31 beads (diameter = 1mm) is approximatively the same as the one in contact with the solution in polystyrene or polypropylene wells when filled with 100 μL solution. mAb adsorption to the hydrophobic surfaces was quantified by protein G-HRP or protein A-HRP binding using an enzymatic read-out with tetramethylbenzidine (TMB) and H 2 O 2 . ELISA results obtained with both HRP constructs gave very similar results (Supplementary Figure 3b). The results represent mean values and their respective standard deviations obtained from a minimum of 8 independent replicate wells. mAbA, B and C, concentrated at 30 g.L -1 , 0.7g.L -1 and 3g.L -1 , respectively, were mixed with different concentrations of PS80 (0, 0.05, 0.1 and 0.2 g.L -1 ) before filling the wells. The plates were filled with 100 μL protein-surfactant solution per well and shaken at 900 rpm (Heidolph Titramax vibrating platform microplate shaker) for 15 minutes. Then each well was emptied and washed 4 times with 200 µL PS80 at 0.2g.L -1 . The wells were then filled with 100µL protein G-or protein A-HRP at 250µg.L -1 containing 0.2g.L -1 PS80 and shaken at 900 rpm for 20 minutes. Four new washing steps were performed as previously described. A tablet (1mg)

Results

PS80 and mAbA adsorption differs on polystyrene and on hexadecane surfaces

We compared the protective effect of PS80 against mAbA adsorption on two hydrophobic surfaces using two complementary surface-sensitive techniques, SPRi and QCM-d. A selfassembled hexadecane (C16) monolayer was chosen as a model hydrophobic surface (WCA = 103 ± 2 °) and polystyrene (PS) (WCA = 89 ± 4°) as an example of a plastic polymer. Goldcoated prisms and QCM sensors were functionalized with C16 and PS as described in the experimental section or directly purchased.

After stabilization of the SPRi or QCM-d signals in buffer, PS80 was injected at 0.2 g.L -1 on both surfaces followed by an injection of buffer allowing to estimate the amount of irreversibly adsorbed surfactant to the surfaces (Figure 1a andb). The results show that PS80 remains adsorbed on both surfaces, with = 5 ± 1.5 RU and = -7.5 ± 0.5 Hz on C16 and = 6.5 ± 2.5 RU and = -11.5 ± 1.5 Hz on polystyrene, measured by SPRi and QCM-d, respectively (Table I). Both techniques thus confirm an irreversible and slightly higher surface adsorption of PS80 on PS than on C16.

Subsequently, mAbA was injected at 30g.L -1 on both surfaces until the signal was stabilized and then desorbed using Bf1x. The reflectivity signals (Figure 1a) show that mAbA remains less adsorbed on PS than on C16 surfaces pre-incubated with PS80, with reflectivity differences equal to 2 ± 2 RU ( -) and 11 ± 0.5 RU ( -), respectively (Table I). Likewise, the level of mAbA adsorption measured by QCM-d on PS is much lower than on C16, when pre-adsorbed with PS80, with frequency differences equal to -3.5 ± 3 Hz ( -and -18 ± 0.5 Hz ( -, respectively.

We conclude that, after a pre-treatment of PS and C16 with PS80, slightly more surfactant remains adsorbed on PS, and mAbA adsorbs significantly less on PS pre-coated with PS80 than on C16 pre-coated in the same way.

Quantification of PS80 protective efficacy on polystyrene and hexadecane surfaces for the formulated mAb

We then developed a protocol to confirm the surface-dependent protection efficacy of PS80 by comparing the amount of surface-adsorbed mAbA in the presence (0.2 g. L -1 , PS80 concentration used in the formulated mAbA) and absence of surfactant on PS and C16functionalized sensors. To do this, we used protein G (PtG), known to bind specifically to the Fc region of IgGs, to measure the amount of surface-adsorbed mAbA. In order to test whether mAb surface adsorption could alter the accessibility for PtG binding sites, we compared the readouts of PtA-and PtG-HRP constructs in an ELISA assay on polystyrene.

Supplementary Figure 3b shows that both HRP constructs have equal performances, indicating that mAbA surface adsorption is unlikely to affect binding site accessibility for PtG.

The PtG reflectivity and frequency signals were measured first on surfaces precoated with PS80 alone at 0.2 g.L -1 to take into account non-specific PtG adsorption (R 0 ) (Figure 2a, Supplementary Figure 1 and Supplementary Table I). These signals were very low, demonstrating that PtG is a valid marker allowing to specifically quantify mAbA surface adsorption. We then measured the PtG reflectivity and frequency signals associated to the surface adsorption of mAbA (30g.L -1 ) without surfactant (R 1 ). We considered R 1 as the value representing the maximal mAbA adsorption level on the surfaces (Figure 2b, Supplementary Figure 1 and Supplementary Table I). A comparison of the respective adsorption levels of mAbA on C16 and PS shows that reflectivity data (SPRi) indicate twice as much mAbA adsorption on PS than on C16 while the frequency data (QCMd) indicate similar adsorbed amounts on both surfaces (Supplementary Table I). This could be due to different PS densities on the spin-coated QCMd sensor and the PS-thiol functionalized SPR prism.

Surface characterisation by XPS (Supplementary Figure 2) indeed confirms the density difference of the PS functionalisations on both sensors. For C16 surfaces, obtained by the same thiol functionalisation protocol on both SPRi and QCMd sensors, the surface densities are similar (Supplementary Figure 2).

To quantify the protective efficacy of the surfactant, we finally measured the PtG reflectivity and frequency signals obtained on the surfaces after adsorption of mAbA (30g.L -1 ) in the presence of 0.2 g.L -1 PS80 (formulation conditions) (R 2 ) (Figure 2c, Supplementary Figure 1 and Supplementary Table I). The amount of mAbA specifically adsorbed to the surfaces was quantified using the measured PtG signals and expressed as (R 2 -R 0 ) /R 1 . The results

show that, on PS, PS80 can limit mAbA adsorption to 46% (by SPRi) and to 33% (by QCM-d), compared to the mAbA adsorption level in the absence of PS80 (Figure 2d). On the C16 surface, however, the protective efficacy of PS80 is very low and mAbA adsorption can only be reduced to 98% (by SPRi) and 91% (by QCM-d), respectively (Figure 2d). These results prove that, PS80 protects mAbA more effectively against adsorption on polystyrene (PS) than on a hexadecane (C16) surface.

Surfactant protection of mAbs depends on the material surface

Conflict of Interest Statement

The authors declare no conflicts of interest.

Author contributions

GL developed the methodology and did the experimental work together with AM; VF and SH provided the molecules for the study; VF, SH, FB and MW are responsible for the supervision, the conceptualization, the formal analysis and the validation; GL and MW prepared the manuscript; VF and SH together with FB and MW secured the funding.

Supplementary Materials

Supplementary Figure 1 Supplementary polystyrene-functionalized sensor. The PtG is injected after: a, an adsorption of PS80 at 0.2g.L -1 (F 0 ) followed by a buffer (B) wash, b, an adsorption of mAbA (A) at 30g.L -1 without surfactant (F 1 ) followed by a buffer (B) wash and c, an adsorption of mAbA at 30g.L -1 with PS80 at 0.2g.L -1 (formulation condition, F)) (F 2 ) followed by a buffer (B) wash.

Supplementary Table I PtG reflectivity and frequency values measured on hexadecane