Post-translational Modifications of Recombinant Proteins: Significance for Biopharmaceuticals

The production of recombinant therapeutic proteins is one of the fastest growing sectors of the pharmaceutical industry, particularly monoclonal antibodies and Fc-fusion proteins. Currently, mammalian cells are the dominant production system for these proteins because they can perform complex post-translational modifications that are often required for efficient secretion, drug efficacy, and stability. These protein modifications include misfolding and aggregation, oxidation of methionine, deamidation of asparagine and glutamine, variable glycosylation, and proteolysis. Such modifications not only pose challenges for accurate and consistent bioprocessing, but also may have consequences for the patient in that incorrect modifications and aggregation can lead to an immune response to the therapeutic protein. This mini-review describes examples analytical and preventative advances in the fields of protein oxidation, deamidation, misfolding and aggregation (glycosylation is covered in other articles in this issue). The feasibility of partially replacing traditional analytical methods such as peptide mapping with high-throughput screens and their use in clone and media selection are evaluated. This review also discusses how further technical advances could improve the manufacturability, potency, and safety of biotherapeutics.     

Review physical and chemical aspects of stabilization of compounds in silk

The challenge of stabilization of small molecules and proteins has received considerable interest. The biological activity of small molecules can be lost as a consequence of chemical modifications, while protein activity may be lost due to chemical or structural degradation, such as a change in macromolecular conformation or aggregation. In these cases, stabilization is required to preserve therapeutic and bioactivity efficacy and safety. In addition to use in therapeutic applications, strategies to stabilize small molecules and proteins also have applications in industrial processes, diagnostics, and consumer products like food and cosmetics. Traditionally, therapeutic drug formulation efforts have focused on maintaining stability during product preparation and storage. However, with growing interest in the fields of encapsulation, tissue engineering, and controlled release drug delivery systems, new stabilization challenges are being addressed; the compounds or protein of interest must be stabilized during: (1) fabrication of the protein or small molecule-loaded carrier, (2) device storage, and (3) for the duration of intended release needs in vitro or in vivo. We review common mechanisms of compound degradation for small molecules and proteins during biomaterial preparation (including tissue engineering scaffolds and drug delivery systems), storage, and in vivo implantation. We also review the physical and chemical aspects of polymer-based stabilization approaches, with a particular focus on the stabilizing properties of silk fibroin biomaterials.     

Improved methods for targeting epigenetic reader domains of acetylated and methylated lysine

Responsible for interpreting histone post-translational modifications, epigenetic reader proteins have emerged as novel therapeutic targets for a wide range of diseases. Chemical probes have been critical in enabling target validation studies and have led to translational advances in cancer and inflammation-related pathologies. Here, we present the most recently reported probes of reader proteins that recognize acylated and methylated lysine. We will discuss challenges associated with achieving potent antagonism of reader domains and review ongoing efforts to overcome these hurdles, focusing on targeting strategies including the use of peptidomimetic ligands, allosteric modulators, and protein degraders.     

Fluorescence complementation: an emerging tool for biological research

Numerous technologies based on utilizing fluorescent proteins have been developed for biological research, and fluorescence complementation (FC) is a recent application for visualization of molecular events in living cells and organisms. Currently, ten fluorescent proteins have been demonstrated to support FC. Over the past five years, FC-based technologies have been developed to visualize a variety of molecular events, such as protein-protein interactions, post-translational modifications, protein folding, conformational changes, RNA-protein interactions, mRNA localization and DNA hybridization. In addition, FC has also been used for drug discovery. These applications are providing fascinating insights into many biological processes. Here, we review the principles and applications of FC technologies, discuss their current challenges and examine prospects for future advances.     

Novel therapeutic strategies for the treatment of protein-misfolding diseases

Most proteins in the cell adopt a compact, globular fold that determines their stability and function. Partial protein unfolding under conditions of cellular stress results in the exposure of hydrophobic regions normally buried in the interior of the native structure. Interactions involving the exposed hydrophobic surfaces of misfolded protein conformers lead to the formation of toxic aggregates, including oligomers, protofibrils and amyloid fibrils. A significant number of human disorders (e.g. Alzheimer disease, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis and type II diabetes) are characterised by protein misfolding and aggregation. Over the past five years, outstanding progress has been made in the development of therapeutic strategies targeting these diseases. Three promising approaches include: (1) inhibiting protein aggregation with peptides or small molecules identified via structure-based drug design or high-throughput screening; (2) interfering with post-translational modifications that stimulate protein misfolding and aggregation; and (3) upregulating molecular chaperones or aggregate-clearance mechanisms. Ultimately, drug combinations that capitalise on more than one therapeutic strategy will constitute the most effective treatment for patients with these devastating illnesses.     

Protein misfolding and aggregation

Interest in the problem of protein misfolding and aggregation has exploded in recent years for two reasons: (1) the sharp rise in the number and volume of therapeutic proteins produced commercially and (2) the recognition of the central role of protein aggregates in degenerative diseases. The systematic study of protein aggregation presents major challenges to both the experimentalist and the theoretician. Much of the work retains an empirical flavor due to the experimental complexities; the sensitivity of protein aggregation to the slightest change in protein amino acid composition, solvent properties, or protein concentration; and the lack of robust theoretical models of misfolding and aggregation. Novel experimental and computational approaches are being developed, and we anticipate substantial progress will be made in the near future. Several presentations describing the latest advances in protein misfolding and aggregation were given at the American Chemical Society meeting (BIOT division) held in September, 2006 in San Francisco.     

Understanding and controlling amyloid aggregation with chirality

Amyloid aggregation and human disease are inextricably linked. Examples include Alzheimer disease, Parkinson disease, and type II diabetes. While seminal advances on the mechanistic understanding of these diseases have been made over the last decades, controlling amyloid fibril formation still represents a challenge, and it is a subject of active research. In this regard, chiral modifications have increasingly been proved to offer a particularly well-suited approach toward accessing to previously unknown aggregation pathways and to provide with novel insights on the biological mechanisms of action of amyloidogenic peptides and proteins. Here, we summarize recent advances on how the use of mirror-image peptides/proteins and d-amino acid incorporations have helped modulate amyloid aggregation, offered new mechanistic tools to study cellular interactions, and allowed us to identify key positions within the peptide/protein sequence that influence amyloid fibril growth and toxicity.     

Arraying proteins by cell-free synthesis

Recent advances in life science have led to great motivation for the development of protein arrays to study functions of genome-encoded proteins. While traditional cell-based methods have been commonly used for generating protein arrays, they are usually a time-consuming process with a number of technical challenges. Cell-free protein synthesis offers an attractive system for making protein arrays, not only does it rapidly converts the genetic information into functional proteins without the need for DNA cloning, but also presents a flexible environment amenable to production of folded proteins or proteins with defined modifications. Recent advancements have made it possible to rapidly generate protein arrays from PCR DNA templates through parallel on-chip protein synthesis. This article reviews current cell-free protein array technologies and their proteomic applications.     

Inhibiting protein aggregation with nanomaterials: The underlying mechanisms and impact factors

Protein aggregation is correlated with the onset and progression of protein misfolding diseases (PMDs). Inhibiting the generation of toxic aggregates of misfolded proteins has been proposed as a therapeutic approach for PMDs. Due to their unique properties, nanomaterials have been extensively investigated for their ability to inhibit protein aggregation and have shown great potential in the diagnosis and treatment of PMDs. However, the precise mechanisms by which nanomaterials interact with amyloidogenic proteins and the factors influencing these interactions remain poorly understood. Consequently, developing a rational design strategy for nanomaterials that target specific proteins in PMDs has been challenging. In this review, we elucidate the effects of nanomaterials on protein aggregation and describe the mechanisms through which nanomaterials interfere with protein aggregation. The major factors impacting protein-nanomaterial interaction such as size, charge, concentration, surface modification and morphology that can be rationally addressed to achieve the desired effects of nanomaterials on protein aggregation are summarized. The prospects and challenges to the clinical application of nanomaterials for the treatment of PMDs are also discussed.     

A screening tool for therapeutic monoclonal antibodies: Identifying the most stable protein and its best formulation based on thioflavin T binding

The lack of a fast selection method to identify the most stable protein is one of the major challenges for developing successful therapeutic protein formulations more rapidly. The swift and accurate detection of small amounts of aggregates is another problem since aggregates may trigger an immunological response and the aggregation decreases the biological activity of the antibody. Here we present an alternative method for initial screening of the aggregation propensity of proteins, using monoclonal antibodies (mAb) as an example and thioflavin T (ThT) binding. The major advantage of ThT binding is the short duration of testing compared with size-exclusion chromatography (SEC) measurements that can take 6 months or more even under accelerated conditions. The tendency to aggregate of each therapeutic human mAb probed with the ThT assay, together with SEC, is employed to formulate the ranking of mAb aggregation. ThT binding can determine the propensity of proteins to aggregate in a few days, illustrating that ThT binding would be a valuable screening tool.     

Posttranslational modification of therapeutic proteins in plants

Plants have emerged as an alternative to current systems for the production of therapeutic proteins. The advantages of plants for the low-cost and large-scale production of safe and biologically active mammalian proteins have been documented recently. A major advantage of transgenic plants over production systems that are based on yeast or Escherichia coli is their ability to perform most of the posttranslational modifications (PTMs) that are required for the bioactivity and pharmacokinetics of recombinant therapeutic proteins. Furthermore, recent advances in the control of PTMs in transgenic plants have made it possible for plants to perform, at least to some extent, human-like modifications of recombinant proteins. Hence, plants have become a suitable alternative to animal cell factories for the production of therapeutic proteins.     

High hydrostatic pressure as a tool to study protein aggregation and amyloidosis

Aggregation of proteins is a serious problem, affecting both industrial production of proteins and human health. Despite recent advances in the theories and experimental techniques available to address understanding of protein aggregation processes, mechanisms of aggregate formation have proved challenging to study. This is in part because the typical irreversibility of protein aggregation processes at atmospheric conditions complicates analysis of their kinetics and thermodynamics. Because high hydrostatic pressures act to disfavor the hydrophobic and electrostatic interactions that cause protein aggregation, studies conducted under high hydrostatic pressures may allow protein aggregates to be formed reversibly, enabling thermodynamic and kinetic parameters to be measured in greater detail. Although application of high hydrostatic pressures to protein aggregation problems is rather recent, a growing literature, reviewed herein, suggests that high pressure may be a useful tool for both understanding protein aggregation and reversing it in industrial applications.     

Emerging roles of O-glycosylation in regulating protein aggregation,phase separation,and functions

Protein O-glycosylation is widely identified in various proteins involved in diverse biological processes. Recent studies have demonstrated that O-glycosylation plays crucial and multifaceted roles in modulating protein amyloid aggregation and liquid–liquid phase separation (LLPS) under physiological conditions. Dysregulation of these processes is closely associated with human diseases such as neurodegenerative diseases (NDs) and cancers. In this review, we first summarize the distinct roles of O-glycosylation in regulating pathological aggregation of different amyloid proteins related to NDs and elaborate the underlying mechanisms of how O-glycosylation modulates protein aggregation kinetics, induces new aggregated structures, and mediates the pathogenesis of amyloid aggregates under diseased conditions. Furthermore, we introduce recent discoveries on O-GlcNAc-mediated regulation of synaptic LLPS and phase separation potency of low-complexity domain-enriched proteins. Finally, we identify challenges in future research and highlight the potential for developing new therapeutic strategies of NDs by targeting protein O-glycosylation.     

Protein arginine methylation promotes therapeutic resistance in human pancreatic cancer

Pancreatic cancer is a lethal disease with limited treatment options for cure. A high degree of intrinsic and acquired therapeutic resistance may result from cellular alterations in genes and proteins involved in drug transportation and metabolism, or from the influences of cancer microenvironment. Mechanistic basis for therapeutic resistance remains unclear and should profoundly impact our ability to understand pancreatic cancer pathogenesis and its effective clinical management. Recent evidences have indicated the importance of epigenetic changes in pancreatic cancer, including posttranslational modifications of proteins. We will review new knowledge on protein arginine methylation and its consequential contribution to therapeutic resistance of pancreatic cancer, underlying molecular mechanism, and clinical application of potential strategies of its reversal.     

Protein post-translational modifications: A key factor in colorectal cancer resistance mechanisms

Colorectal cancer (CRC) is one of the leading causes of cancer-related death. Despite advances in treatment, drug resistance remains a critical impediment. Post-translational modifications (PTMs) regulate protein stability, localization, and activity, impacting vital cellular processes. Recent research has highlighted the essential role of PTMs in the development of CRC resistance. This review summarizes recent advancements in understanding PTMs' roles in CRC resistance, focusing on the latest discoveries. We discuss the functional impact of PTMs on signaling pathways and molecules involved in CRC resistance, progress in drug development, and potential therapeutic targets. We also summarize the primary enrichment methods for PTMs. Finally, we discuss current challenges and future directions, including the need for more comprehensive PTM analysis methods and PTM-targeted therapies. This review identifies potential therapeutic interventions for addressing medication resistance in CRC, proposes prospective therapeutic options, and gives an overview of the function of PTMs in CRC resistance.     

Preparative expression of secreted proteins in bacteria: status report and future prospects

The expression of heterologous secreted proteins in Escherichia coli is widely employed for laboratory and preparative purposes. Thanks to advances in expression technologies over the past 25 years, many mammalian proteins can now be produced routinely in secreted form with yields in the gram/litre scale. Nonetheless, ensuring efficient secretion across the inner membrane, and preventing proteolytic degradation, incorrect disulfide-bond formation and aggregation into periplasmic inclusion bodies, frequently presents significant challenges. Recent advances in the understanding of the periplasmic folding quality control system are leading to new strategies to facilitate the expression of heterologous secreted proteins. In parallel, protein design and directed evolution approaches are beginning to be exploited for engineering of the cellular protein folding machinery to achieve further improvements in protein expression.     

Post-aggregation Oxidation of Mutant Huntingtin Controls the Interactions between Aggregates

Aggregation of protein molecules is a pathological hallmark of many neurodegenerative diseases. Abnormal modifications have often been observed in the aggregated proteins, supporting the aggregation mechanism regulated by post-translational modifications on proteins. Modifications are in general assumed to occur in soluble proteins before aggregation, but actually it remains quite obscure when proteins are modified in the course of the aggregation. Here we focus upon aggregation of huntingtin (HTT), which causes a neurodegenerative disorder, Huntington disease, and we show that oxidation of a methionine residue in HTT occurs in vitro and also in vivo. Copper ions as well as added hydrogen peroxide are found to oxidize the methionine residue, but notably, this oxidative modification occurs only in the aggregated HTT but not in the soluble state. Furthermore, the methionine oxidation creates additional interactions among HTT aggregates and alters overall morphologies of the aggregates. We thus reveal that protein aggregates can be a target of oxidative modifications and propose that such a “post-aggregation” modification is a relevant factor to regulate properties of protein aggregates.     

Emerging trends in plasma-free manufacturing of recombinant protein therapeutics expressed in mammalian cells

Mammalian cells are the expression system of choice for therapeutic proteins, especially those requiring complex post-translational modifications. Traditionally, these cells are grown in medium supplemented with serum and other animal- or human-derived components to support viability and productivity. Such proteins are also typically added as excipients and stabilizers in the final drug formulation. However, the transmission of hepatitis B in the 1970s and of hepatitis C and HIV in the 1980s through plasma-derived factor VIII concentrates had catastrophic consequences for hemophilia patients. Thus, due to regulatory concerns about the inherent potential for transmission of infectious agents as well as the heterogeneity and lack of reliability of the serum supply, a trend has emerged to eliminate the use of plasma-derived additives in the production and formulation of recombinant protein therapeutics. This practice began with products used in the treatment of hemophilia and is progressively expanding throughout the entire industry. The plasma-free method of producing recombinant therapeutics is accomplished by the use of both cell culture media and final product formulations that do not contain animal- or human-derived additives. A number of recombinant therapeutic proteins for the treatment of several different diseases have been produced by plasma-free processes, with the objective of improving safety by eliminating blood-borne pathogens or by reducing immunogenicity. This review describes the factors that drove the development of plasma-free protein therapeutics and provides examples of advances in manufacturing that have made possible the removal of human and animal-derived products from all steps of recombinant protein production.     

Effect of methylglyoxal modification on stress-induced aggregation of client proteins and their chaperoning by human alphaA-crystallin

alpha-Crystallin prevents protein aggregation under various stress conditions through its chaperone-like properties. Previously, we demonstrated that MGO (methylglyoxal) modification of alphaA-crystallin enhances its chaperone function and thus may affect transparency of the lens. During aging of the lens, not only alphaA-crystallin, but its client proteins are also likely to be modified by MGO. We have investigated the role of MGO modification of four model client proteins (insulin, alpha-lactalbumin, alcohol dehydrogenase and gamma-crystallin) in their aggregation and structure and the ability of human alphaA-crystallin to chaperone them. We found that MGO modification (10-1000 microM) decreased the chemical aggregation of insulin and alpha-lactalbumin and thermal aggregation of alcohol dehydrogenase and gamma-crystallin. Surface hydrophobicity in MGO-modified proteins decreased slightly relative to unmodified proteins. HPLC and MS analyses revealed argpyrimidine and hydroimidazolone in MGO-modified client proteins. The degree of chaperoning by alphaA-crystallin towards MGO-modified and unmodified client proteins was similar. Co-modification of client proteins and alphaA-crystallin by MGO completely inhibited stress-induced aggregation of client proteins. Our results indicate that minor modifications of client proteins and alphaA-crystallin by MGO might prevent protein aggregation and thus help maintain transparency of the aging lens.     

Adapting protein sequences for optimized therapeutic efficacy

Therapeutic proteins alleviate disease pathology by supplementing missing or defective native proteins, sequestering superfluous proteins, or by acting through designed non-natural mechanisms. Although therapeutic proteins often have the same amino acid sequence as their native counterpart, their maturation paths from expression to the site of physiological activity are inherently different, and optimizing protein sequences for properties that 100s of millions of years of evolution did not need to address presents an opportunity to develop better biological treatments. Because therapeutic proteins are inherently non-natural entities, optimization for their desired function should be considered analogous to that of small molecule drug candidates, which are optimized through expansive combinatorial variation by the medicinal chemist. Here, we review recent successes and challenges of protein engineering for optimized therapeutic efficacy.