Aggregation of Therapeutic Proteins

Wei Wang, Christopher J. Roberts, Wei Wang, Christopher J. Roberts

• Publisher: Wiley
• Year: 2010
• Print ISBN: 9780470411964
• eBook ISBN: 9781118043585
• Edition: 1
• Topic: Medicine
• Subtopic: Pharmacology

This book gives pharmaceutical scientists an up-to-date resource on protein aggregation and its consequences, and available methods to control or slow down the aggregation process. While significant progress has been made in the past decade, the current understanding of protein aggregation and its consequences is still immature. Prevention or even moderate inhibition of protein aggregation has been mostly experimental. The knowledge in this book can greatly help pharmaceutical scientists in the development of therapeutic proteins, and also instigate further scientific investigations in this area. This book fills such a need by providing an overview on the causes, consequences, characterization, and control of the aggregation of therapeutic proteins.

CHAPTER 1

Fundamental Structures and Behaviors of Proteins

JENNIFER S. LAURENCE and C. RUSSELL MIDDAUGH

Protein aggregation has been increasingly recognized as a problem limiting the efficacy and shelf life of protein therapeutics and as an indicator and cause of numerous disease states. Elucidating the molecular mechanisms behind aggregation has become a central focus of investigation in order to improve therapeutics and to understand the relationship between aggregate formation and cellular toxicity in protein misfolding diseases. Innovations in analysis techniques, particularly of solid-state materials, and computational molecular modeling approaches have provided higher resolution information about the structure of aggregates as well as key insights into the mechanisms of aggregate formation. These breakthroughs, coupled with understanding gained from solution experiments and biological systems, have just begun to enable strategies to combat aggregation, including the design and evaluation of peptides and small molecules that inhibit the growth or that facilitate the dissociation of aggregates. This chapter describes the fundamental properties of proteins and the current understanding of underlying mechanisms that influence native folding and the formation of aggregates.

1.1 THE PROBLEM OF PROTEIN AGGREGATION

Protein aggregation has significant influence in the pathology, onset, and progression of most, if not all, misfolding diseases. Over 40 human diseases have been linked to aggregation of a specific protein, including hemoglobin in sickle cell anemia, the widely recognized Aβ peptides in Alzheimer’s disease, the PrP prion protein in Creutzfeldt–Jakob’s and related diseases, expanded polyglutamine tracts in Huntington’s disease, amylin-induced β-cell death in diabetes, and α-synuclein in Parkinson’s disease.¹ Moreover, studies of non-disease-associated proteins in vitro show that aggregates and amyloid fibers can be induced to occur from almost any protein, suggesting it is a ubiquitous phenomenon reflecting a common mechanism.² Therapeutic proteins used to treat various diseases can also produce ill effects when aggregates are present, in some cases contributing to amyloid plaque formation in vivo.^3,4 Aggregates have been observed to form in therapeutic proteins during purification and storage, and the administration of proteins containing aggregates has been shown to stimulate immune responses, causing effects ranging from mild skin irritation to anaphylaxis.^5,6 As such, major efforts are underway to stabilize therapeutic proteins against aggregation. Thus, the goal of understanding the fundamental properties of proteins that contribute to aggregation and the mechanisms by which they aggregate is of critical importance for determining how to prevent and treat numerous diseases.

In vivo protein aggregation appears to be an ever-present problem caused by thermal fluctuations and chemical changes that disrupt the physical structure of these delicate molecules. Consequently, cells have evolved several mechanisms by which they prevent aggregates from interfering with normal function.⁷ Improperly folded proteins are removed from cells before they can initiate aggregation by being degraded into smaller peptides via the proteosome or lysosomal enzymes. Alternatively, intracellular proteins can be refolded to their native conformation by interaction with chaperone proteins, which are often expressed at elevated levels in response to thermal (heat shock) or chemical stress. Chaperones bind to hydrophobic patches on misfolded proteins and use an energy-dependent process to alter their conformation, therein providing the protein with additional attempts to find its native fold.⁸ When the capacity of the aforementioned machinery is exceeded, aggregates may form,⁹ as is often observed in recombinant expression systems. As one might expect, coexpression with chaperones can reduce the formation of aggregates in some cases. Chaperones have been demonstrated to affect aggregation not only by improving recovery of soluble protein but also conversely to promote aggregation when present at high levels. When aggregates form in vivo, sequestration mechanisms exist that recognize aggregated species and shuttle them to designated storage locations within the cell, such as the bacterial inclusion body and aggresome or newly discovered IPOD and JUNQ sites in eukaryotic cells.^10,11 When the cellular machinery is overwhelmed by excessive damage to normal proteins or by mutations that generate a less stable form of a protein that accelerates aggregation, disease or death may result. Evidence for this is found in that increased amounts of proteosomal and chaperone proteins are found colocalized with aggregates in these inclusions.

Recombinant expression has become an increasingly important method for producing large amounts of protein for therapeutic and biotechnology applications. Production of recombinant protein is often frustrated by aggregation in the host. Yield can sometimes be improved by decreasing the temperature at which the protein is made or by coexpression with chaperone proteins (e.g., GroEL) to aid folding in vivo and to reduce sequestration to inclusion bodies.⁸ Nonetheless, proteins are often shuttled to inclusion bodies. Aggregated proteins, however, may be folded in vitro from the insoluble state. Single-domain proteins less than 150 residues, which are directed to inclusion bodies, can sometimes be extracted from the solid aggregate and refolded. Denaturing conditions are used to disrupt associations between chains, and the denatured material is diluted into a non-denaturing solution so that it may refold into its native form. When the native form of the protein contains disulfide bonds, folding is carried out under defined redox conditions to facilitate proper disulfide formation. This approach is not very efficient, typically resulting in a substantial fraction of the protein returning to an insoluble state. This observation suggests that proteins may follow different pathways during the course of folding, of which only some are productive. Very limited success has been had using this approach with large, multidomain proteins or those with numerous or more complex posttranslational modifications. The difficulty in refolding these proteins probably derives from increased competition between alternative interactions with those of the native state. These incorrect associations may lead to misfolding when the rate of protein production or the context in which the protein is produced is altered. Addition of chaperones at the dilution step has been used to enhance refolding of proteins that otherwise aggregate. The strategy is also being applied to stabilize purified proteins during storage. Once the active form is purified, proteins are commonly maintained at cold temperatures to restrict their conformational flexibility and to preserve their structural integrity.

Protein folding and unfolding may not follow the same pathway.¹² Some proteins fold and unfold reversibly, yet may accomplish each event using a different approach to overcome transition state barriers. Other proteins require assistance to attain their native conformation but subsequently are quite resistant to unfolding. These observations suggest that a protein’s ability to arrive at its native conformation and to maintain it is not necessarily ruled to the same degree by the same parameters. Proteins are not static and undergo a variety of different types of conformational fluctuations. The range of states sampled is dictated by noncovalent interactions that stabilize the native fold and the effect of external influences like temperature and solution conditions on their interactions. Factors that drive folding influence the stability of the folded form, but coincident interactions that develop as a consequence of the folded state also impact retention of the native fold and help determine the frequency of transitions to partially unfolded conformations and subsequent progression to aggregated states. Once the folded protein is obtained, stabilization against transitions to aggregated states becomes a critical issue. Understanding how to prevent aggregation has primarily been based on empirical studies. The next objective is to elucidate the mechanisms that determine how transitions from the native ensemble promote aggregation.

1.1.1 Structural Features of Proteins

Proteins are linear polymers. Their primary structure is composed of 20 naturally occurring amino acids having diverse chemical properties. The amino acids are typically alpha amino acids and have L chirality. Each is joined by a peptide bond, which has a planar character that restricts the conformational freedom of the backbone of the polypeptide chain. As such, common structural features are observed among folded proteins, most broadly falling into the categories of alpha helix, beta sheet, turns, and disordered regions. These first three secondary structural elements are developed as a result of hydrogen bonding interactions that involve atoms from within the polypeptide backbone. Disordered regions lack such hydrogen bonding patterns. Alpha helices are almost always right handed and have a register in which the carbonyl oxygen from residue i forms a hydrogen bond with the amide proton four residues to its C-terminus (i + 4). The alpha helix contains 3.6 residues per turn, and due to the slight offset in vertical alignment, a secondary twist develops with elongation. Rarely, a short 3₁₀-helix has been observed to form, in which i to i + 3 bonding occurs. The even rarer π-helix utilizes i to i + 5 bonding. Due to favored dihedral backbone angles and steric constraints, alpha is the most favorable helical organization. It often occurs in isolation, whereas the other two forms are found only in small segments in folded proteins in which the structural context provides stabilization for these less favorable structural elements. Beta-sheet structure is also favorable and can arise from a parallel or antiparallel alignment of the strands. The pattern of hydrogen bonding differs between these two sheet organizations. Antiparallel strands may form from contiguous or discontinuous primary structure, but parallel association necessarily occurs between sequences that have intervening secondary structural elements.

Contiguous stretches of repeated H-bonds stabilize each structural element and help compact the polymer within local regions of the sequence. This limits the number of possible arrangements between distal segments, which also facilitates the establishment of a preferred three-dimensional conformation (tertiary structure). Three-dimensional coalescence into a compact state generally relies on interactions between amino acid side chains. The diverse chemical composition of the side chains produces both attractive and repulsive forces, and the native configuration derives from the formation of the most energetically favorable associations between distal moieties that stabilize the packed arrangement within a folded domain. The majority of interactions that contribute to protein folding are noncovalent, but covalent bonding between the thiol-containing moieties of cysteine residues may occur to generate a disulfide bond under oxidizing conditions. Disulfide bonds are common among secreted proteins, where they often greatly enhance the ability of the protein to resist unfolding.

Globular domain folds are classified into families that range from all helical to mixed alpha-beta to all beta composition. Regardless of the domain architecture, separate polypeptide chains can further associate into homotypic or heterotypic oligomers to yield a quaternary structure. Individual subunits in an oligomeric complex can simply physically associate based on surface complementation, but they may also be covalently tethered. Covalent attachment ensures close proximity and is most often accomplished through intermolecular disulfide bonds.

Noncovalent association between subunits vary in affinity based on the same principles that dictate protein folding, and several modes of interaction have been described, including lock and key, induced fit, and preexisting equilibrium/conformational selection mechanisms. Lock-and-key binding implies the structure is unaltered by the binding event. Induced fit models suggest that the protein adopts a new state in response to binding to its partner, whereas conformational selection indicates that in the associated complex, an existing state is stabilized. Analogous modes of interaction may also apply to protein–protein associations that pertain to physical aggregation. A relatively new area of investigation has demonstrated the diversity of conformations that can result from the same sequence. For example, natively disordered proteins have been suggested to adopt distinct conformations in different contexts to perform discrete functions. Recent studies also reveal that globular proteins can maintain more than one unique stable conformation. Moreover, crystal structures are often reported for the same protein in distinct oligomeric states depending on solution conditions. These findings suggest that preferences in the conformation of a protein, even those below the current limit of detection, are influenced by the context in which the protein resides.

1.1.2 Structural Features of Protein Aggregates

Macroscopic attributes of aggregates have been described from data acquired using a variety of microscopy techniques. In the most general terms, the morphological features commonly observed are usually categorized as amorphous or fibrillar. Amorphous aggregates are present in inclusion bodies in vivo and often emerge during the course of processing and storing protein samples. Amorphous aggregates lack long-range order and are often opaque if they are not soluble. They were originally thought to contain completely unfolded material held together by random associations between hydrophobic residues. The hypothesis that amorphous aggregates lack discernable structure was derived from (1) early observation that harsh denaturants like sodium dodecyl sulfate (SDS), urea, and guanidine-HCl (Gdn) are required to resolubilize proteins from inclusion bodies; and (2) a lack of data concerning structural features owing to the fact that amorphous materials often scatter light, interfering with the spectroscopic analyses typically used to characterize structure. In contrast, some aggregates retain native activity, and the active form of some proteins can only be recovered from inclusion bodies when mild denaturing conditions are used, whereas aggressive denaturation leads to an inability to refold the protein (e.g., hGH),¹³ suggesting that the species present in the inclusion body contain elements of native structure that facilitate refolding. Additionally, staining methods have been used to reveal the presence of regular structure within amorphous aggregates. Staining of some aggregates and not others by dyes like Congo red (CR) and thioflavin T (ThT) suggests that proteins directed to inclusion bodies contain at least some ordered structural elements common to fibrillar aggregates.^14,15 Many aggregates have increased beta-sheet content and diminished alpha helicity compared to the native state, which is presumably developed through intermolecular contacts. Although the molecular organization of amorphous aggregates remains rather coarsely described overall, the Weliky lab recently provided evidence for native-like structure within amorphous aggregates. Their examination of influenza virus hemagglutinin within an inclusion body using solid-state nuclear magnetic resonance (NMR) indicates the presence of a substantial retention of helical structure. The residues that form helices in the aggregate correspond well to those present in the native conformation observed in the available crystal structure.¹⁶ This result, combined with the fact that amorphous aggregates from inclusion bodies bind CR and ThT, suggests that aggregates are composed of a combination of native- and fibrillar-like structures.

Fibrillar aggregates are commonly, but not always, associated with the formation of amyloid plaques and are named for their long, thin, fibrous shape. Great diversity in the diameter, length, and in...

Table of contents

• Cover
• Half title page
• Title page
• Copyright page
• Dedication
• PREFACE
• CONTRIBUTORS
• CHAPTER 1 Fundamental Structures and Behaviors of Proteins
• CHAPTER 2 Protein Aggregation Pathways, Kinetics, and Thermodynamics
• CHAPTER 3 Identification and Impact of Aggregation-Prone Regions in Proteins and Therapeutic Monoclonal Antibodies
• CHAPTER 4 External Factors Affecting Protein Aggregation
• CHAPTER 5 Experimental Detection and Characterization of Protein Aggregates
• CHAPTER 6 Approaches to Control Protein Aggregation during Bulk Production
• CHAPTER 7 Protein Aggregation and Particle Formation: Effects of Formulation, Interfaces, and Drug Product Manufacturing Operations
• CHAPTER 8 Approaches to Managing Protein Aggregation in Product Development
• CHAPTER 9 Case Studies Involving Protein Aggregation
• CHAPTER 10 Aggregation and Immunogenicity of Therapeutic Proteins
• CHAPTER 11 Regulatory Perspective on Aggregates as a Product Quality Attribute
• Index
• Color Plates