Protein modification during antiviral heat bioprocessing

Heat treatment is routinely used in the preparation of therapeutic protein biopharmaceuticals as a means of viral inactivation. However, in undertaking virucidal heat treatments, a balance must be found between the bioprocessing conditions, virus kill, and the maintenance of protein integrity. In this study, we utilize a simple model protein, hen egg-white lysozyme, to investigate the relationship between antiviral bioprocess conditions (protein formulation and temperature) and the extent and type of protein modification. A variety of industrially relevant wet- and dry-heat treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Although there was no evidence of lysozyme aggregation or crosslinking during any of the heat treatments, using liquid chromatography-electrospray ionization-mass spectroscopy (LC-ESI-MS) and peptide mapping we show that protein modifications do occur with increasingly harsh heat treatment. Modifications were predominantly found after wet-heat treatment, the major covalent modification of lysozyme under these conditions being glycation of Lys(97), by either glucose or fructose derived from hydrolyzed sucrose. The extent of sucrose hydrolysis was itself dependent on both the duration of heat treatment and formulation composition. Advanced glycation end products (AGEs) and additional unidentified products were also present in protein samples subjected to extended heat treatment. AGEs were derived primarily from initial glycation by fructose and not glucose. These findings have implications for the improvement of bioprocesses to ensure protein product quality.     

Protein modification during anti-viral heat-treatment bioprocessing of factor VIII concentrates, factor IX concentrates, and model proteins in the presence of sucrose.

To ensure the optimal safety of plasma derived and new generation recombinant proteins, heat treatment is customarily applied in the manufacturing of such biopharmaceuticals as a means of viral inactivation. In subjecting proteins to anti-viral heat-treatment it is necessary to use high concentrations of thermostabilizing excipients to prevent protein damage, and it is therefore imperative that the correct balance between bioprocessing conditions, maintenance of protein integrity and virus kill is found. In this study we have utilized model proteins (lysozyme, fetuin, and human serum albumin) and plasma-derived therapeutic proteins (factor VIII and factor IX) to investigate the protein modifications that occur during anti-viral heat treatment. Specifically, we investigated the relationship between bioprocessing conditions and the type and extent of protein modification under a variety of industrially relevant wet and lyophilized heat treatments using sucrose as a thermostabilizing agent. Heat treatment led to the formation of disulfide crosslinks and aggregates in proteins containing free cysteine residues. Terminal oligosaccharide sialic acid residues were hydrolyzed from the glycan moieties of glycoproteins during anti-viral heat treatment. Heat treatment promoted sucrose hydrolysis to yield glucose and fructose, leading, in turn, to the glycation of lysine amino groups in those proteins containing di-lysine motifs. During extended hear treatments, 1,2-dicarbonyl type advanced glycation end-products were also formed. Glycation-type modifications were more prevalent in wet heat-treated protein formulations.     

NMR analysis of synthetic human serum albumin alpha-helix 28 identifies structural distortion upon amadori modification

The non-enzymatic reaction between reducing sugars and long-lived proteins in vivo results in the formation of glycation and advanced glycation end products, which alter the properties of proteins including charge, helicity, and their tendency to aggregate. Such protein modifications are linked with various pathologies associated with the general aging process such as Alzheimer disease and the long-term complications of diabetes. Although it has been suggested that glycation and advanced glycation end products altered protein structure and helicity, little structural data and information currently exist on whether or not glycation does indeed influence or change local protein secondary structure. We have addressed this problem using a model helical peptide system containing a di-lysine motif derived from human serum albumin. We have shown that, in the presence of 50 mm glucose and at 37 degrees C, one of the lysine residues in the di-lysine motif within this peptide is preferentially glycated. Using NMR analysis, we have confirmed that the synthetic peptide constituting this helix does indeed form a alpha-helix in solution in the presence of 30% trifluoroethanol. Glycation of the model peptide resulted in the distortion of the alpha-helix, forcing the region of the helix around the site of glycation to adopt a 3(10) helical structure. This is the first reported evidence that glycation can influence or change local protein secondary structure. The implications and biological significance of such structural changes on protein function are discussed.     

Glycation of antibodies: Modification,methods and potential effects on biological functions

Glycation is an important protein modification that could potentially affect bioactivity and molecular stability, and glycation of therapeutic proteins such as monoclonal antibodies should be well characterized. Glycated protein could undergo further degradation into advance glycation end (AGE) products. Here, we review the root cause of glycation during the manufacturing, storage and in vivo circulation of therapeutic antibodies, and the current analytical methods used to detect and characterize glycation and AGEs, including boronate affinity chromatography, charge-based methods, liquid chromatography-mass spectrometry and colorimetric assay. The biological effects of therapeutic protein glycation and AGEs, which ranged from no affect to loss of activity, are also discussed.     

Inhibition of protein glycation and advanced glycation end products by ascorbic acid and other vitamins and nutrients

Nonenzymatic glycation, the reaction of glucose and other reducing sugars with protein, reversibly produces Amadori products and over a long period irreversible advanced glycation end products. In diabetes, these reactions are greatly accelerated and are important in the pathogenesis of diabetic complications.

In vitro glycation was studied with bovine albumin as the model protein. A mixture of 25 mM glucose/fructose was used as the glycating agent. The Amadori product was quantitated by thiobarbituric acid colorimetry after hydrolysis. Advanced glycation end products were measured by their intrinsic fluorescence. A number of vitamins and nutrients were found to be potent inhibitors of both the glycation reaction and the subsequent end products. The nutrients were effective at physiological concentrations and exhibited dose-response relationships. The inhibitors included ascorbic acid, tocopherol, pyridoxal, niacinamide, sodium selenite, selenium yeast, and carnosine. A significant correlation was found between the inhibition of glycation and the inhibition of AGE formation (P < 0.001). One of the nutrients, ascorbic acid, was used in a pilot study. Eighteen normal subjects, 7 college age and 10 middle age, were supplemented with 1,000 mg of ascorbic acid in the form of Re-Natured Vitamin C^® for a period of 4 weeks. Serum protein glycation was decreased an average of 46.8% (P < 0.01). These results underline the importance of nutrition in diabetes and indicate the possibility of therapeutic use of these nutrients for the prevention of diabetic complications.     

Quantification of glycated IgG in CHO supernatants: A practical approach

Post-translational, nonenzymatic glycation of monoclonal antibodies (mAbs) in the presence of reducing sugars (in bioprocesses) is a widely known phenomenon, which affects protein heterogeneity and potentially has an impact on quality, safety, and efficacy of the end product. Quantification of individual glycation levels is compulsory for each mAb therapeutically applied in humans. We therefore propose an analytical method for monitoring glycation levels of mAb products during the bioprocess. This is a useful tool for process-design considerations, especially concerning glucose-feed strategies and temperature as major driving factors of protein glycation. In this study, boronate affinity chromatography (BAC) was optimized for determination of the glycation level of mAbs in supernatants. In fact, the complex matrix found in supernatants is an underlying obstacle to use BAC, but with a simple clean-up step, we found that the elution profile could be significantly improved so that qualitative and quantitative determination could be reached. Complementary analytical methods confirmed the performance quality, including the correctness and specificity of the results. For quantitative determination of mAb glycation in supernatants, we established a calibration procedure for the retained mAb peak, identified as glycated antibody monomers. For this approach, an available fully characterized mAb standard, Humira®, was successfully applied, and continuous monitoring of mAbs across three repetitive fed-batch processes was finally performed. With this practical, novel approach, an insight was obtained into glycation levels during bioprocessing, in conjunction with glucose levels and product titer over time, facilitating efficient process development and batch-consistency monitoring.     

Antiglycation and cell protective actions of metformin and glipizide in erythrocytes and monocytes

Chronic hyperglycaemia causes glycation which subsequently results in the long-term complications of diabetes. Albumin, the major plasma protein is more sensitive to glycation resulting in structural, biological and physiological modifications. The long-term benefits of commonly used anti-diabetic drugs such as metformin and glipizide in diabetic patients are well understood. However, no extensive study has been performed to assess their role in the glycation induced albumin modifications and cellular protection. We carried out the glycation of bovine serum albumin using methylglyoxal as a glycating agent in absence or presence of metformin and glipizide to establish their anti-glycation action. Different glycation markers (fructosamine, carbonyl groups, free thiol groups and β-amyloid aggregation) and protein structural markers (absorption spectroscopy and native-polyacrylamide gel electrophoresis) were examined. Further THP-1 cells (monocytes) and erythrocytes were treated with drugs that were exposed to glycated albumin samples for 24 h, respectively at 37 °C to investigate the cytoprotective actions of drugs against glycation. After the treatment different anti-oxidant indices (catalase, glutathione, superoxide dismutase and nitric oxide), cell viability, lipid peroxidation and erythrocyte hemolysis were determined. Treatment with metformin and glipizide during in vitro albumin glycation significantly reduced the formation of glycation adducts and inhibited structural modifications. They restored the level of antioxidants in THP-1 and erythrocytes cells treated with glycated albumin thus protecting cells. Our results suggested protection mode of albumin glycation through inhibition by metformin and glipizide. Additionally, they exerted inhibitory actions on glycation-induced cellular damage by restoring cellular antioxidant defense.     

Rejuvenated Vintage Tissue Sections Highlight Individual Antigen Fate During Processing and Long-term Storage

Antigen-bearing proteins become progressively unavailable to immunodetection after prolonged storage of routine sections, exposed to a variety of agents, such as moisture, oxygen, and temperature. By proteomic analysis, the antigens are retained in the sections and definitely in the tissue block, pointing to fixation-independent, storage time–dependent protein modifications. Based on previous experience, we hypothesized that a combined exposure to a reducing agent and to chemicals favoring protein conformation changes would reverse the masking in aged sections. Disaccharides, lactose and sucrose, and a surfactant, added to a standard antigen retrieval buffer, reverse the negative changes in aged sections. Furthermore, they provide enhanced access to antigens in freshly cut sections, but not universally, revealing additional factors, besides heat and calcium chelation, required for antigen retrieval of individual proteins:     

Glycation modulates alpha-synuclein fibrillization kinetics: A sweet spot for inhibition

Glycation is a nonenzymatic posttranslational modification (PTM) known to be increased in the brains of hyperglycemic patients. Alpha-synuclein (αSN), a central player in the etiology of Parkinson’s disease, can be glycated at lysine residues, thereby reducing αSN fibril formation in vitro and modulating αSN aggregation in cells. However, the molecular basis for these effects is unclear. To elucidate this, we investigated the aggregation of αSN modified by eight glycating agents, namely the dicarbonyl compound methylglyoxal (MGO) and the sugars ribose, fructose, mannose, glucose, galactose, sucrose, and lactose. We found that MGO and ribose modify αSN to the greatest extent, and these glycation products are the most efficient inhibitors of fibril formation. We show glycation primarily inhibits elongation rather than nucleation of αSN and has only a modest effect on the level of oligomerization. Furthermore, glycated αSN is not significantly incorporated into fibrils. For both MGO and ribose, we discovered that a level of ∼5 modifications per αSN is optimal for inhibition of elongation. The remaining sugars showed a weak but optimal inhibition at ∼2 modifications per αSN. We propose that this optimal level balances the affinity for the growing ends of the fibril (which decreases with the extent of modification) with the ability to block incorporation of subsequent αSN subunits (which increases with modification). Our results are not only relevant for other αSN PTMs but also for understanding PTMs affecting other fibrillogenic proteins and may thus open novel avenues for therapeutic intervention in protein aggregation disorders.     

Histone H1 as a Reporter Protein to Investigate Glycation in Bacteria

Nonenzymatic glycosylation (glycation) of proteins is a multistage chemical process starting as a condensation reaction between reducing sugars and primary amino groups (mainly from the side chains of Lis and Arg) and ending up with formation of complex heterocyclic compounds called advanced glycation end products (AGEs). For a long time, glycation has been attributed to the long-lived eukaryotes (including in humans) only. In a recent study, we showed that glycation also occurs in bacteria. The present study aims to prove that bacterial cytoplasm contains soluble glycating compounds. To this end, Lis/Arg-rich histone H1 isolated from rat liver was treated with deproteinized Escherichia coli cytoplasm through a dialysis membrane. This treatment leads to accumulation of AGEs as well as to a remarkable degradation of the reporter protein on storage at 4°C. Our results indicate also that glycation can be inhibited by acetylsalicylic acid (aspirin), thiamine (vitamin B1), and pyridoxine (vitamin B6).     

Effect of Maltodextrin Dextrose Equivalent on Electrospinnability and Glycation Reaction of Blends with Pea Protein Isolate

Compared to commonly applied wet and dry heating procedures, a combination of electrospinning and heat treatment can facilitate glycation of proteins with reducing polysaccharides. This study investigates how the amount of reducing carbonyl groups (i.e. dextrose equivalent, DE) of different maltodextrins influences electrospinnability and subsequent glycation in blends with pea protein isolate (PPI). In the first step of the study, maltodextrin-PPI dispersions were electrospun. The concentrations of PPI and maltodextrin DE 2 were kept constant in the aqueous spinning dispersion. The addition of 0.05 or 0.1 g/mL maltodextrin DE 12 or 21 slightly affected the electrical conductivity and dynamic viscosity of the spinning dispersions, however, fiber production rate and morphology were dominated by the presence of maltodextrin DE 2 (0.8 g/mL). In the second step of the study, fibers were heated (60 °C, 75% rel. Humidity, 0–24 h). SDS-PAGE analysis and the measurement of free amino groups confirmed the covalent attachment of maltodextrin carbonyl groups to free amino groups of PPI. The fastest glycation and the lowest relative amount of free amino groups (49.70 ± 6.54%) after 24 h heating was measured for the fibers with the highest amount of reducing carbonyl groups. The fibers with the lowest amount of reducing carbonyl groups showed no significant (p < 0.05) decrease of free amino groups after heat treatment. The results suggest that within the boundaries of electrospinnability, the degree of glycation can be adjusted by varying the amount of reducing carbonyl groups in the fibers.

     

Modifications of therapeutic proteins: challenges and prospects

The production of therapeutic proteins is one of the fastest growing sectors of the pharmaceutical industry. However, most proteins used in drug therapy require complex post-translational modifications for efficient secretion, drug efficacy and stability. Common protein modifications include variable glycosylation, misfolding and aggregation, oxidation of methionine, deamidation of asparagine and glutamine, and proteolysis. These modifications not only pose challenges for accurate and consistent bioprocessing, but also may have consequences for the patient in that incorrect modifications or aggregation may lead to an immune response to the protein therapeutic. This review provides examples of analytical and preventative advances that have been devised to meet these challenges, and insights into how further advances can improve the efficiency and safety in manufacturing recombinant proteins.     

Rates and impact of human antibody glycation in vivo

Glycation of immunoglobulin G (IgG) can result from incubation with a reducing sugar in vitro or during circulation in vivo. Upon injection of a recombinantly produced human therapeutic IgG into humans, changes in the glycation levels could be observed as a function of circulation time. Mass changes on the individual IgG polypeptide chains as the results of glycation were determined using reversed-phase liquid chromatography/mass spectrometry. Changes to the light and heavy chains were low but easily detectable at 0.00092 and 0.0021 glucose (Glc) additions per chain per day, respectively. Levels of glycation found on the Fc portion of IgG isolated from healthy subjects, using a similar analytical approach, were on average 0.045 Glc molecules per fragment. In vivo glycation rates could be approximated in vitro by modeling the physiological glycation reaction with a simplified incubation containing physiological Glc concentrations, pH and temperature but with a high concentration of a single purified IgG. To test the impact of glycation on IgG function, highly glycated IgG1 and IgG2 were prepared containing on average 42-49 Glc molecules per IgG. Binding to FcγIIIa receptors, neonatal Fc receptor or protein A was similar or identical to the non-glycated IgG controls. Although the modifications were well distributed throughout the protein sequence, and at high enough levels to affect the elution position by size-exclusion chromatography, no changes in the tested Fc functions were observed.     

Modern-day challenges in therapeutic protein production

The market for therapeutic proteins is on the rise, plagued by several challenges related to production amounts and costs. Solutions to these problems are widely thought to come from academia, governments and production companies. This conference aimed to bring experts in the industry together under one roof, in order to demystify several novel technologies in therapeutic protein development. Key topics included analytical tools for protein stability and ligand interactions, measurement of protein aggregates as small as 30 nm and reducing production costs, just to name a few. The need to eliminate protein aggregates early during bioprocessing was emphasized. Finally, several companies presented novel technologies related to therapeutic protein development.     

Prevention of lens protein glycation by taurine

Modifications in lens protein structure and function due to nonenzymic glycosylation and oxidation have been suggested to play a significant role in the pathogenesis of sugar and senile cataracts. The glycation reaction involves an initial Schiff base formation between the protein NH₂ groups and the carbonyl group of a reducing sugar. The Schiff base then undergoes several structural modifications, via some oxidative reactions involving oxygen free radicals. Hence certain endogenous tissue components that may inhibit the formation of protein-sugar adduct formation may have a sparing effect against the cataractogenic effects of sugars and reactive oxygen. The eye lens is endowed with significant concentration of taurine, a sulfonated amino acid, and its precursor hypotaurine. It is hypothesized that taurine and hypotaurine may have this purported function of protecting the lens proteins against glycation and subsequent denaturation, in addition to their other functions. The results presented herein suggest that these compounds are indeed capable of protecting glycation competitively by forming Schiff bases with sugar carbonyls, and thereby preventing the glycation of lens proteins per se. In addition, they appear to prevent oxidative damage by scavenging hydroxyl radicals. This was apparent by their preventive effect against the formation of the thiobarbituric acid reactive material generated from deoxy-ribose, when the later was exposed to hydroxyl radicals generated by the action of xanthine oxidase on hypoxanthine in presence of iron.     

A new tool for monoclonal antibody analysis

Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, and terminal cyclization. The modifications in different domains of the antibody molecule can result in different biological consequences. Therefore, characterization and routine monitoring of domain-specific modifications are essential to ensure the quality of the therapeutic antibody products. For this purpose, a rapid and informative methodology was developed to examine the heterogeneity of individual domains in mAb products. A recently discovered endopeptidase, IdeS, cleaves heavy chains below the hinge region, producing F(ab')₂ and Fc fragments. Following reduction of disulfide bonds, three antibody domains (LC, Fd, and Fc/2) can be released for further characterization. Subsequent analyses by liquid chromatography/mass spectrometry, capillary isoelectric focusing, and glycan mapping enable domain-specific profiling of oxidation, charge heterogeneity, and glycoform distribution. When coupled with reversed phase chromatography, the unique chromatographic profile of each molecule offers a simple strategy for an identity test, which is an important formal test for biopharmaceutical quality control purposes. This methodology is demonstrated for a number of IgGs of different subclasses (IgG1, IgG2, IgG4), as well as an Fc fusion protein. The presented technique provides a convenient platform approach for scientific and formal therapeutic mAb product characterization. It can also be applied in regulated drug substance batch release and stability testing of antibody and Fc fusion protein products, in particular for identity and routine monitoring of domain-specific modifications.     

A new tool for monoclonal antibody analysis: Application of IdeS proteolysis in IgG domain-specific characterization

Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, and terminal cyclization. The modifications in different domains of the antibody molecule can result in different biological consequences. Therefore, characterization and routine monitoring of domain-specific modifications are essential to ensure the quality of the therapeutic antibody products. For this purpose, a rapid and informative methodology was developed to examine the heterogeneity of individual domains in mAb products. A recently discovered endopeptidase, IdeS, cleaves heavy chains below the hinge region, producing F(ab')₂ and Fc fragments. Following reduction of disulfide bonds, three antibody domains (LC, Fd, and Fc/2) can be released for further characterization. Subsequent analyses by liquid chromatography/mass spectrometry, capillary isoelectric focusing, and glycan mapping enable domain-specific profiling of oxidation, charge heterogeneity, and glycoform distribution. When coupled with reversed phase chromatography, the unique chromatographic profile of each molecule offers a simple strategy for an identity test, which is an important formal test for biopharmaceutical quality control purposes. This methodology is demonstrated for a number of IgGs of different subclasses (IgG1, IgG2, IgG4), as well as an Fc fusion protein. The presented technique provides a convenient platform approach for scientific and formal therapeutic mAb product characterization. It can also be applied in regulated drug substance batch release and stability testing of antibody and Fc fusion protein products, in particular for identity and routine monitoring of domain-specific modifications.     

Formation of advanced glycation end (AGE) products in diabetes: Prevention by pyruvate and a-keto glutarate

Glycation of proteins and their subsequent structural and functional modifications have been ascribed to play a prominent role in the pathogenesis of several secondary complications of diabetes, such as cataract and retinopathy. In addition, it plays a role in the generalized ageing process as well. Investigations have been conducted to explore the possibility of preventing the above process by use of pyruvate and a-keto glutarate as representatives of physiologically compatible keto acids. The results demonstrate that both these compounds are effective in preventing the initial glycation reaction as well as the formation of AGE products. Both these compounds also inhibit the generation of high molecular weight aggregates associated with cataract formation. Mechanistically, the preventive effects appear to be due to (1) competitive inhibition of glycation by the keto acids and (2) the antioxidant (radical scavenging) properties of these compounds. The results are hence considered usefu l from the point of view of developing these and other keto acid derivatives as pharmacological agents useful in preventing glycation related protein changes and consequent tissue pathological manifestations.     

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.     

Amides are novel protein modifications formed by physiological sugars

The Maillard reaction, or nonenzymatic browning, proceeds in vivo, and the resulting protein modifications (advanced glycation end products) have been associated with various pathologies. Despite intensive research only very few structures have been established in vivo. We report here for the first time N(6)-[2-[(5-amino-5-carboxypentyl)amino]-2-oxoethyl]lysine (GOLA) and N(6)-glycoloyllysine (GALA) as prototypes for novel amide protein modifications produced by reducing sugars. Their identity was confirmed by independent synthesis and coupled liquid chromatography/mass spectrometry. Model reactions with N(alpha)-t-butoxycarbonyl-lysine showed that glyoxal and glycolaldehyde are immediate precursors, and reaction pathways are directly linked to N(epsilon)-carboxymethyllysine via glyoxal-imine structures. GOLA, the amide cross-link, and 1,3-bis(5-amino-5-carboxypentyl)imidazolium salt (GOLD), the imidazolium cross-link, share a common intermediate. The ratio of GOLA to GOLD is greater when glyoxal levels are low at constant lysine concentrations. GOLA and GALA formation from the Amadori product of glucose and lysine depends directly upon oxidation. With the advanced glycation end product inhibitors aminoguanidine and pyridoxamine we were able to dissect oxidative fragmentation of the Amadori product as a second mechanism of GOLA formation exactly coinciding with N(epsilon)-carboxymethyllysine synthesis. In contrast, the formation of GALA appears to depend solely upon glyoxal-imines. After enzymatic hydrolysis GOLA was found at 66 pmol/mg of brunescent lens protein. This suggests amide protein modifications as important markers of pathophysiological processes.