Basic mechanisms leading to in vitro protein aggregation

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Protein folding and stability: to aggregate or not to aggregate?

Antibodies are versatile tools for the treatment of several diseases and constitute nowadays the fastest growing class of human drugs. Yet, aggregation of antibodies during both production and post-production stages gives rise to undesired side-e ects (immunogenicity of aggregates, toxicity) that currently restrain progress in the eld.
This rst introductory chapter is focused on the basic mechanisms that are at the origin of protein aggregation. A particular attention is paid to proteins of therapeutic interest, notably antibodies. The chapter also describes some of the main conventional strategies adopted to prevent aggregation, both during storage under potentially stressful conditions and during refolding procedures. Motivations of the work and the necessity to nd other stabilising approaches are introduced.

Preamble: about the development of therapeutic anti-bodies and practical issues

Since the end of the 1990s, the downward trend of therapeutic innovation has become apparent [1, 2]. Costs associated to the production of new drugs soar and leave no room for improvisation. The numerous patent expiries of blockbusters drugs in the forthcoming years contributes to erce competition of generic drugs, sold on average 60% cheaper than their branded equivalents. Harsher regulatory requirements, in particular in terms of innocuousness, and scandals in the pharmaceutical industry also fuel the increase of production costs of drugs. Accordingly, pharmaceutical groups tend to progressively abandon the research of drugs based on proprietary molecule libraries and chemical modi cations, and turn now to the rational design of biomolecules. Whereas chemistry and search for \lead » small molecules (screening) was at the center of developments in the 1980s, by 2004 some 50% of the projects underway in drug companies were based on biotechnology [survey of the USA Biotechnology Industry Organization], a trend that was boosted by the allowance to patent genes and genetically-modi ed organisms (GMO) since the early 1980s. More personalized treatments are in the pipeline, seeking at rapid diagnosis of a disease (through the identi cation of speci c biological markers) and subsequent prescription of the best suited treatment for a given patient. The word \theranostic » has been used to describe this coupling between therapy and diagnosis. Vaccines, hormones, enzymes or antibodies are highly versatile (bioengineerable) and promising tools in this context: these drugs are designated as \biopharmaceuticals » or \biologics », namely drugs derived from biological sources.
Among therapeutic proteins, antibodies play an increasingly important role in the treatment of viral infections (including HIV) [3], autoimmune disorders [4], cancers [5{7] and several other human diseases [8]. This preamble aims to introduce some of the milestones that marked out the development of these biopharmaceuticals, as well as give some economic considerations and issues that currently restrain progress in the eld. We will particularly justify the need to control protein folding and stability.

Antibody-based therapies

More than a century has passed since the discovery of agents { that were soon to be called \antibodies » { capable of conferring immunity against diseases such as diphtheria that were until then incurable. Nowadays, antibodies are routinely used to treat cancer and other scourges of modern society. Motivations for the use of antibodies can be found in their structure and biological role.

Antibody structure and biological role: an overview

Antibodies are glycoproteins (i.e. proteins that contain oligosaccharides chains covalently attached to the polypeptide backbone) belonging to the superfamily of \immunoglobu-lins » (Ig). All proteins of this family share a structural motif called \immunoglobulin domain » [9], which structure will be analysed more in detail in Chapter 4 and Chapter 5. In a simplistic view, several Ig domains assemble (through non-covalent interactions and disul de bridges) to form one antibody molecule that adopts a distorted \Y » shape (see Figure 1.1). In mammals, antibodies are classi ed into ve main classes, referred to as \isotypes » (IgG, IgA, IgM, IgE and IgD). Each class has a distinct structure and biological activity but they all possess the common \Y »-shaped element [9].
Immunoglobulins G (IgG) constitute the most abundant isotype found in normal human serum (accounting for ∼ 70-85% of seric antibodies) [8, 10]. These proteins of ca. 150 kDa molecular weight consist of a heterotetramer of two identical light chains (MW ∼ 25 kDa) and two identical heavy chains (MW ∼ 50 kDa) held together by disulfide bridges (Figure 1.1) [11]. Each IgG heavy chain contains a variable domain (VH) and three conserved domains (CH1, CH2 and CH3), while an IgG light chain is composed of a single variable domain (VL) and a single constant domain (CL) [9]. Enzymatic cleavage can be used to separate the two identical Fab arms of the “Y”-shaped protein (fragment antigen-binding) from the Fc fragment (fragment crystallizable) [12]. These three fragments are connected via the hinge region that confers a certain flexibility to IgG. The two antigen-binding sites (or paratopes) are located at the very ends of each arm of the “Y”-shaped molecule (N-terminal domains, purple regions in Figure 1.1), and contain particularly diverse strands of amino acids that form 6 hypervariable loops, also called complementarity-determining regions (CDR), which confers a high specificity to each IgG.
IgG are involved in the protection of organisms against pathogens or toxins by neutralizing them and inducing their elimination. Lymphocyte B (or B-cells) are able to produce a seemingly unlimited number of IgG that can be highly specific for a given antigen recognized as foreign by the organism. IgG can function by different modes of action, as schematically illustrated on Figure 1.2. Upon binding to cell receptors, IgG can in particular recruit proteins from the complement system, phagocytes and cytotoxic cells (schematically recruited and activated via the Fc region), which eventually leads to the destruction (lysis or phagocyosis) of the targeted element. A detailed mechanism of action of IgG is beyond the scope of this introduction and can be found in several reviews [4, 7].
Antibody-based therapies aim to eliminate or neutralize pathogens or diseased cells. Upon administration, therapeutic antibodies are expected to specifically bind to their target – which is, in ∼ 70% of cases, a surface protein (membrane receptors, glycoproteins, adhesion molecules or viral capsid proteins for instance) – subsequently inducing the elimination of the addressed element, without affecting healthy tissues (see Figure 1.3).
Figure 1.2: Schematic illustration of the main biological roles of IgG. (a) Ligand neutralization: IgG bind a toxin or a virus thus preventing it from activating their cognate cell receptors and penetrating cells. (b) Receptor blockade: IgG act as antagonists by inhibiting a cell receptor. (c) Complement pathway activation: IgG activate complement-mediated lysis and opsonization. (d) Opsonization and phagocytosis: the pathogen is digested by phagocytes that are recruited by IgG (via the Fc fragment) and opsonins.
(e) Signalling induction: IgG induce cascade reactions (active signals) that alter cellular fates. (f) Receptor downregulation: binding of cell surface receptors by IgG result in their internalization (thus limit the receptors that can be activated). (g) Antibody-mediated cell cytotoxicity: effector cells recruited by IgG secrete proteins that contributes to the lysis of the cell.

Historical and economical considerations

The development of therapeutic antibodies has been greatly facilitated when production of monodisperse monoclonal antibodies (mAbs), i.e. antibodies targeted against one specific antigen, became possible, after the work of K¨ohler and Milstein who, back in the 1970s, developed methods for the isolation of monoclonal antibodies [13].
Advances in genetic engineering and molecular biology in the 1990s made available humanization of mAbs, paving the way to their use as specifically targeted therapeutic agents in humans (see Timeline on Figure 1.6) [14, 15]. Problems of anti-antibodies immune responses were partly solved with the use of chimeric antibodies [16] (murine variable part and human constant region), then humanized antibodies [17] (murine amino acids of the variable region replaced by human residues, except in the hypervariable regions [18, 19]) and eventually fully human antibodies. Therapeutic antibodies are nowadays used routinely in the treatment of severe diseases: more than 40 therapeutic antibodies and their derivatives have been approved by the FDA in the past 25 years and 338 are currently in clinical trials [2013 PhRMA report on biologics in development].
Therapeutic mAbs now constitute the fastest growing class of human drugs [20]. Soon after their regulatory approval, antibody products have already reached breathtaking sales: Humira, for instance, has now become the first worldwide best-selling drug (> $10 billion worldwide sales in 2013) only 12 years after its commercialization [Medscape]. As an illustration, Figure 1.4 plots the worldwide sales of the top 10 best-selling drugs in 2013. Seven of these drugs are biopharmaceuticals, six of which are antibody-based products. These top six antibodies account for a total annual sale in excess of $48 billion, which represents ca. 8% of the global market for pharmaceuticals. Due to the almost infinite combinations of sequences that can be designed, financial investments will certainly continue at high rate in the upcoming years. The global market of recombinant antibodies is expected to growth by more than 12% in 2013-2017 [Reuters].

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Other formats: antibody-drug conjugates and antibody fragments

Most therapeutic mAbs today appear not to be sufficiently potent to be efficient on their own and are hence often used in combination with conventional chemical drugs (“drug cocktail”) [21]. For cancer therapy, efforts are now more and more directed towards the coupling of cytotoxic drugs onto monoclonal antibodies, with the aim of increasing tumour selectivity and efficacy of chemical drugs with minimal side effects on healthy tissues. These chemically-modified antibodies have been termed antibody-drug conjugates (ADC). By now, two ADC have already gained regulatory approval from the FDA (for a recent review, see Chari et al. [21]).
Other antibody formats are also largely being developed. In the past few years, the variety of antibody structures has indeed been widely extended. Advances in recombinant DNA technology has enabled the design of fusion proteins (such as protein-Fc fusions, called immunoadhesins) and fragments of antibodies that have been further assembled to obtain very diverse nano-constructs. Figure 1.5 lists some of the already existing antibody-based constructs. Amongst them, single-chain Fv fragments (scFvs) have
become interesting building blocks of antibody fragments. These artificial constructs are made of the variable regions of the heavy and light chains of an IgG that have been linked together via a peptide linker, thus retain the high specificity and affinity of antibodies and can multimerize depending on the chain length of the peptide linker to form dimeric, trimeric or tetrameric forms, conferring at will multi-specificity to the constructs.
Antibody fragments offer advantages over complete IgG. Their smaller size in particu-lar increases their diffusion rate into tissues, as well as their clearance rate, making these tools interesting markers of tumoral tissues for in vivo imaging (fragments coupled with fluorescent reporters or radio-isotopes). Chapter 4 will focus on scFvs currently developed for therapeutic imaging. A complete review of antibody fragments in development is beyond the scope of this introduction. The interested reader shall find more details in reviews [22], [23] and [24].

A practical bottleneck: aggregation of therapeutic proteins

Origin of antibody aggregation

Therapeutic mAbs { as well as their fragments and other therapeutic proteins { face practical issues that hamper their present development. One of the main bottlenecks is undoubtedly their in vitro aggregation, which is an ubiquitous hurdle during biopharma-ceuticals’ expression and puri cation, storage, shipping and administration.
Aggregation can manifest upon production of recombinant proteins Recombinant proteins over-expressed in bacteria are hardly produced under their native form but tend to be expressed into in cellulo insoluble aggregates called inclusion bodies (IBs) [25]. Albeit usually highly homogeneous, which may facilitate protein puri cation, inclusion bodies are often regarded as a bottleneck since refolding procedures are needed, after solubilization of the IBs, to obtain active proteins. Frequently used chaotropic agents to solubilize IBs include denaturants, such as urea and guanidinium chloride (GndCl), that break intra- and inter-molecular interactions, but also ionic detergents (such as sodium dodecylsulfate) [26]. Reductants may also be added to disperse aggregates of proteins containing disul de briges. Refolding of IBs is not a straightforward process, and often requires many steps of handling (exchange of bu er conditions, addition and removal of additives, immobilisation/release of the protein which is both time-consuming and expensive) [25]. In addition, it may lead to the formation of insoluble protein aggregates, in particular when working with arti cial antibody fragments that have not been screened to optimize their solubility.
In order to gain regulatory approval, mAbs need to be puri ed after production. These puri cation treatments involve several steps, typically including protein A chromatography (for capture of the product and removal of impurities), viral inactivation (performed at low pH of 3-4), cation/anion exchange chromatography as well as ultra- ltration (to sterilize the samples). Because of the far-from-physiological conditions used, all this steps are critical for IgG aggregation.
Aggregation can manifest upon long-term storage/shipping/administration As opposed to common chemical (molecular) drugs, that are only subjected to potential chemical degradation upon storage, therapeutic proteins also face physical degrada-tion issues. Environmental stresses (temperature excursions, pH changes, agitation, freeze/thaw, adsorption to air-liquid or solid-liquid interfaces, shear, light exposure…) easily lead to the formation of protein aggregates because of partial unfolding (see details in Section 1.2). In addition, because of the relatively high dose required for e cacy, formulations tend to be prepared at high concentrations (typically 10-50 mg.mL-1) which further enhances self-association.

Side-e ects of aggregation of therapeutic proteins

Aggregates must be avoided in therapeutic formulations. First of all, aggregates often exhibit a lower activity than the monomeric protein, therefore requiring multiple injection at a higher dose to reach the correct dose for treatment e cacy. Second, from a practical point of view, aggregates are associated to increased production costs, since they require more puri cation steps and expensive optimization of formulations (based on trial-and-error approaches). Third, aggregates have been shown to cause both toxicity and immune responses upon administration to patients [27{31], which mainly manifests through the formation of binding or neutralizing antibodies (anti-drug antibodies, ADA) that can also decrease the activity of the therapeutic protein.
The ability of protein aggregates to elicit unwanted immune responses against the monomeric form of the protein of interest has been known for more than half a century, but little is known about the particularities that make protein aggregates potent inducers of the immune system. It has been shown that, depending on the size, structure, amount or solubility, not all aggregates are immunogenic [32]. According to current knowledge, high-order multimeric protein aggregates containing native-like molecules with repetitive epitope presentation seem to be the most immunogenic type of aggregates.
Overall, aggregation of therapeutic proteins fosters decreased production yields (i.e. increased associated costs), loss of activity and is a key factor in causing adverse events associated with immunogenicity in the clinic. To avoid formation of aggregates, it is hence crucial to control the stability of IgG and to understand what the origin of such aggregation is. We will see in the next section that the presence of partially folded (resp. unfolded) protein intermediates generated during refolding steps (resp. under environmental stresses) is critical in the formation of aggregates.

Table of contents :

General Introduction
I Proteins in solution: from folding and stability issues to the design of synthetic chaperones 
1 Protein folding and stability: to aggregate or not to aggregate?
1.1 Preamble: about the development of therapeutic antibodies and practical issues
1.2 Basic mechanisms leading to in vitro protein aggregation
1.3 Conventional strategies to limit in vitro protein aggregation
2 Towards the design of polymer-based and colloidal articial chaperones
2.1 In vivo folding and aggregation: role and mode of action of natural chaperones
2.2 En route to the design of articial chaperones
2.3 Amphiphilic polyelectrolyte as promising articial chaperones: protein binding, influence on conformational stability and on aggregation of proteins
2.4 Our project: study of model poly(sodium acrylate) derivatives as articial chaperones for antibodies and their derivatives
II Chemical refolding of proteins in the presence of amphiphilic polyelectrolytes 
3 Renaturation of a model enzyme with amphiphilic poly(acrylate) chaperones
3.1 Bovine carbonic anhydrase B: a model enzyme to study aggregation during refolding
3.2 Keeping CAB soluble with poly(acrylate) derivatives
3.3 Polymers allow recovery of a native-like and active protein
3.4 Conclusion: discussion on the role of Coulomb and hydrophobic associations
4 Chemical refolding of scFv fragments with poly(acrylate) derivatives
4.1 Preamble: structure, use and folding of scFv
4.2 Size of scFv during refolding
4.3 Do polymers aect the secondary structure of scFv?
4.4 Conclusive remarks and perspectives
III Amphiphilic polyelectrolytes for the stabilization of IgG 
5 Stabilization of IgG during thermal stress in the presence of poly(acrylate) derivatives
5.1 Motivation to study IgG stability
5.2 IgG:polymer complexes at room temperature
5.3 Evidence of association between polymers and heat-stressed IgG
5.4 Impact of poly(acrylate) derivatives on IgG aggregation upon thermal stress
5.5 Conclusion
Conclusion and perspectives 
A Materials and methods
B Experimental techniques
C Supplementary data on CAB refolding assisted with PAA derivatives
D Supplementary data on scFv refolding assisted with PAA derivatives
E Supplementary data on thermally-induced IgG aggregation in the presence of PAA derivatives


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