Forced degradation of recombinant monoclonal antibodies: A practical guide

Forced degradation studies have become integral to the development of recombinant monoclonal antibody therapeutics by serving a variety of objectives from early stage manufacturability evaluation to supporting comparability assessments both pre- and post- marketing approval. This review summarizes the regulatory guidance scattered throughout different documents to highlight the expectations from various agencies such as the Food and Drug Administration and European Medicines Agency. The various purposes for forced degradation studies, commonly used conditions and the major degradation pathways under each condition are also discussed.     

The role of formulation in insulin comparability assessments.

The concept of comparability can be applied when changes are made to manufacturing processes for biotechnology products subsequent to pivotal clinical trial studies. For many process changes, comparability can be demonstrated based entirely on relevant in vitro data provided that a detailed knowledge of the process/product exists, suitable analytical methodology is employed, and historical data are available for the assessment. Insulin provides an excellent model system to illustrate many important considerations when dealing with comparability exercises for biotechnology products. The physicochemical properties of insulin demonstrate the numerous chemical reactions and physical transformations that are exclusive to proteins. These properties are heavily influenced by formulation conditions and must be carefully evaluated when process changes are made. In addition, physical and chemical testing performed on representative formulations can provide valuable insight when assessing the comparability between pre- and post-change materials. This paper reviews our experience with manufacturing changes involving insulin emphasizing the important role of formulation in the comparability exercise for protein biopharmaceuticals.     

Impact of IgG Fc‐Oligosaccharides on Recombinant Monoclonal Antibody Structure,Stability, Safety,and Efficacy

Glycosylation of the conserved asparagine residue in the CH2 domain is the most common posttranslational modification of recombinant monoclonal antibodies. Ideally, a consistent oligosaccharide profile should be maintained from early clinical material to commercial material for the development of recombinant monoclonal therapeutics, though variation in the profile is a typical result of process changes. The risk of oligosaccharide variation posed to further development is required to be thoroughly evaluated based on its impact on antibody structure, stability, efficacy and safety. The variation should be controlled within a range so that there is no detrimental impact on safety and efficacy and thus allowing the use of early phase safety and efficacy data to support project advancement to later phase. This review article focuses on the current scientific understanding of the commonly observed oligosaccharides found in recombinant monoclonal antibodies and their impact on structure, stability and biological functions, which are the basis to evaluate safety and efficacy. It also provides a brief discussion on critical quality attribute (CQA) assessment with regard to oligosaccharides based on the mechanism of action (MOA). © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1173–1181, 2017     

Comparability testing of a humanized monoclonal antibody (Synagis) to support cell line stability, process validation, and scale-up for manufacturing.

Biochemical and functional testing of a humanized monoclonal antibody directed against Respiratory Syncytial Virus (Synagis) has been performed to evaluate cell line stability, support process validation, and to demonstrate "comparability" during the course of process development. Using a variety of analytical methods, product manufactured at different sites and in bioreactors from 20 litres to 10,000 litres was shown to be biochemically and functionally equivalent. The biochemical testing for microheterogeneity found on Synagis included evaluation of changes in post-translational modifications such as deamidation, truncation, and carbohydrate structure. Studies were also performed to support cell line stability assessment and cell culture process validation. Cell culture conditions were deliberately varied in an attempt to determine if this would have an impact on the microheterogeneity of the product. In these studies Synagis was produced from cells cultured beyond the population doublings achieved at the maximum manufacturing scale, under conditions of low glucose, and using harvest times outside of the historical manufacturing operating range. Results showed that there was a different pattern of glycosylation during the early stages of bioreactor culture. No other changes in microheterogeneity were apparent for the other culture conditions studied. In summary, comparability assessment demonstrated that the Synagis manufacturing process is robust and consistent resulting in a predictable and reproducible monoclonal antibody product.     

Charge variants in IgG1

Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7-9.1 and was composed of about 20% acidic variants, 12% basic variants, and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic, and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences, and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs, and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity, or the PK properties in rats.     

Analytical lessons learned from selected therapeutic protein drug comparability studies

The successful implementation of process and product changes for a therapeutic protein drug, both during clinical development and after commercialization, requires a detailed evaluation of their impact on the protein's structure and biological functionality. This analysis is called a comparability exercise and includes a data driven assessment of biochemical equivalence and biological characterization using a cadre of analytical methodologies. This review focuses on describing analytical results and lessons learned from selected published therapeutic protein comparability case studies both for bulk drug substance and final drug product. An overview of the currently available analytical methodologies typically used is presented as well as a discussion of new emerging analytical techniques. The potential utility of several novel analytical approaches to comparability studies is discussed including distribution and stability of protein drugs in vivo, and enhanced evaluation of higher-order protein structure in actual formulations using hydrogen/deuterium exchange mass spectrometry, two-dimensional nuclear magnetic resonance fingerprinting or empirical phase diagrams. In addition, new methods for detecting and characterizing protein aggregates and particles are presented as these degradants are of current industry-wide concern. The critical role that analytical methodologies play in elucidating the structure–function relationships for therapeutic protein products during the overall assessment of comparability is discussed.     

Monoclonal antibodies as probes of conformational changes in protein-engineered cytochrome c

Determination of the nature of the antigen-antibody complex has always been the ultimate goal of three-dimensional epitope mapping studies. Various strategies for epitope mapping have been employed which include comparative binding studies with peptide fragments of antigens, binding studies with evolutionarily related proteins, chemical modifications of epitopes, and protection of epitopes from chemical modification or proteolysis by antibody shielding. In this study we report the use of protein engineering to modify residues in horse cytochrome c that are in or near the epitopes of four monoclonal antibodies specific for this protein. The results demonstrate not only that site-specific changes in the antigen binding site dramatically affect antibody binding, but, more importantly, that some of the site-specific changes cause local and long-range perturbations in structure that are detected by monoclonal antibody binding at other surfaces of the antigen. These findings emphasize the role of native conformation in the stabilization of the interaction between protein antigens and high affinity monoclonal antibodies. Furthermore, the results demonstrate that monoclonal antibodies are more sensitive probes of changes in conformation brought about by protein engineering than low resolution spectroscopic methods such as circular dichroism, where similar spectra are observed for all the analogues. These findings suggest a role for monoclonal antibodies in detecting conformational changes invoked by nonconservative amino acid substitutions or substitutions of evolutionarily conserved residues in protein-engineered or recombinant proteins.     

Side-by-side comparability of batch and continuous downstream for the production of monoclonal antibodies

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.     

Controlling trisulfide modification in recombinant monoclonal antibody produced in fed-batch cell culture

Molecular heterogeneity was detected in a recombinant monoclonal antibody (IgG1 mAb) due to the presence of a trisulfide linkage generated by the post‐translational insertion of a sulfur atom into disulfide bonds at the heavy–heavy and heavy–light junctions. This molecular heterogeneity had no observable effect on antibody function. Nevertheless, to minimize the heterogeneity of the IgG1 mAb from run‐to‐run, an understanding of the impact of cell culture process conditions on trisulfide versus disulfide linkage formation was desirable. To investigate variables that might impact trisulfide formation, cell culture parameters were varied in bench‐scale bioreactor studies. Trisulfide analysis of the samples from these runs revealed that the trisulfide content in the bond between heavy and light chains varied considerably from <1% to 39%. Optimizing the culture duration and feeding strategy resulted in more consistent trisulfide levels. Cysteine concentration in the feed medium had a direct correlation with the trisulfide level in the product. Systematic studies revealed that cysteine in the feed and the bioreactor media was contributing hydrogen sulfide which reacted with the IgG1 mAb in the supernatant leading to the insertion of sulfur atom and formation of a trisulfide bond. Cysteine feed strategies were developed to control the trisulfide modification in the recombinant monoclonal antibody. Biotechnol. Bioeng. 2012; 109: 2523–2532. © 2012 Wiley Periodicals, Inc.     

Biosimilars advancements: Moving on to the future

Many patents for the first biologicals derived from recombinant technology and, more recently, monoclonal antibodies (mAbs) are expiring. Naturally, biosimilars are becoming an increasingly important area of interest for the pharmaceutical industry worldwide, not only for emergent countries that need to import biologic products. This review shows the evolution of biosimilar development regarding regulatory, manufacturing bioprocess, comparability, and marketing. The regulatory landscape is evolving globally, whereas analytical structure and functional analyses provide the foundation of a biosimilar development program. The challenges to develop and demonstrate biosimilarity should overcome the inherent differences in the bioprocess manufacturing and physicochemical and biological characterization of a biosimilar compared to several lots of the reference product. The implementation of approaches, such as Quality by Design (QbD), will provide products with defined specifications in relation to quality, purity, safety, and efficacy that were not possible when the reference product was developed. Actually, the need to prove comparability to the reference product by the biosimilar industry has increased the knowledge about the product and the production‐process associated by the use of powerful analytical tools. The technological challenges to make copies of biologic products while attending regulatory and market demands are expected to help innovation in the direction of attaining more productive manufacturing processes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1139–1149, 2015     

Worldwide experience with biosimilar development

Limited access for high-quality biologics due to cost of treatment constitutes an unmet medical need in the US and other regions of the world. The term “biosimilar” is used to designate a follow-on biologic that meets extremely high standards for comparability or similarity to the originator biologic drug that is approved for use in the same indications. Use of biosimilar products has already decreased the cost of treatment in many regions of the world and now a regulatory pathway for approval of these products has been established in the US. The Food and Drug Administration (FDA) led the world with the regulatory concept of comparability and the European Medicines Agency (EMA) was the first to apply this to biosimilars. Patents on the more complex biologics, especially monoclonal antibodies, are now beginning to expire and biosimilar versions of these important medicines are in development. The new Biologics Price Competition and Innovation Act (BPCIA) allows the FDA to approve biosimilars and allows the FDA to lead on the formal designation of interchangeability of biosimilars with their reference products. The FDA''s approval of biosimilars is critical to facilitating patient access to high-quality biologic medicines and will allow society to afford the truly innovative molecules currently in the global biopharmaceutical industry''s pipeline.Key words: monoclonal antibodies (mAbs), biosimilars, recombinant biopharmaceuticals     

Heterologous recombinant expression of non-originator NISTmAb

The successful development and regulatory approval of originator and biosimilar therapeutic proteins requires a systems approach to upstream and downstream processing as well as product characterization and quality control. Innovation in process design and control, product characterization strategies, and data integration represent an ecosystem whose concerted advancement may reduce time-to-market and further improve comparability and biosimilarity programs. The biopharmaceutical community has made great strides to this end, yet there currently exists no pre-competitive monoclonal antibody (mAb) expression platform for open innovation. Here, we describe the development and initial expression of an intended copy of the NISTmAb using three non-originator murine cell lines. It was found that, without optimization and in culture flasks, all three cell lines produce approximately 100 mg mAb per liter of culture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, nuclear magnetic resonance spectroscopy, intact mass spectrometry, and surface plasmon resonance were used to demonstrate that the products of all three cell lines embody quality attributes with a sufficient degree of sameness to the NISTmAb Reference Material 8671 to warrant further bioreactor studies, process improvements and optimization. The implications of the work with regard to pre-competitive innovation to support process design and feedback control, comparability and biosimilarity assessments, and process analytical technologies are discussed.     

The art and artifact of GDF9 activity: cumulus expansion and the cumulus expansion-enabling factor

The process of cumulus cell expansion is critical for normal fertility. Oocyte-produced growth and differentiation factor 9 (GDF9) has been thought to play a leading role in this process. Recent studies both support and refute this hypothesis. Central to understanding the physiology of GDF9 is the use of recombinant ligand in in vitro assays. There are several laboratories that currently produce recombinant GDF9 preparations that appear to show variable effects on granulosa cell gene expression and cumulus cell expansion. Several of these studies are reviewed here. Standardization in preparation for recombinant GDF9, as well as a more biochemical analysis of the oocyte-secreted forms of GDF9, may help to resolve the conflicts currently seen in the literature.     

Charge variants in IgG1: Isolation,characterization, in vitro binding properties and pharmacokinetics in rats

Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7–9.1 and was composed of about 20% acidic variants, 12% basic variants and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity or the PK properties in rats.Key words: mAb IgG1, charge heterogeneity, isoelectric point, neonatal Fc receptor (FcRn), pharmacokinetics, potency     

Distinct immunological and biochemical properties of thyroid peroxidase purified from human thyroid glands and recombinant protein produced in insect cells.

The biosynthesis of thyroid hormone from thyroglobulin is catalysed by thyroid peroxidase (TPO), an integral membrane protein. TPO is also a major autoantigen in autoimmune thyroid disease and autoantibodies to TPO are markers for disease activity. Large quantities of purified TPO are essential for elucidating its structure and understanding its role in disease activity. We describe the high yield purification of full-length recombinant human TPO from baculovirus infected insect cells and compare it to purified native TPO from human thyroid glands. In contrast to native human TPO, the human TPO produced in insect cells as a recombinant protein was insoluble and resistant to solubilisation in detergents. Reversible substitution of lysine residues with citraconic anhydride led to increased solubility of the recombinant TPO, allowing high-yield purification by monoclonal antibody chromatography. The purified enzyme preparation was shown to be TPO by its reactivity with monoclonal and polyclonal antibodies by enzyme linked immunosorbent assay and Western blotting. Both the human and recombinant purified TPO preparations also react with sera from patients with autoimmune thyroid disease, although the binding of conformational dependent autoantibodies was considerably lower to the recombinant TPO than to the native TPO. This suggests that the recombinant TPO may differ in some aspects of its tertiary structure. The purified recombinant TPO was devoid of enzyme activity, in contrast to the enzymatically active, purified human TPO preparations. Both preparations contained comparable amounts of haem (R(z)=0.269), but a shift in the Soret band of recombinant TPO (402 nm) from that of natural TPO (409 nm) indicates that the lack of enzymatic activity of the recombinant enzyme may be due to changes in the protein backbone surrounding the haem. Both the purified native and recombinant TPO, under non-denaturing conditions, show evidence of high molecular mass oligomers, although the latter preparation is prone to a greater degree of aggregation. In conclusion, our studies indicate that recombinant TPO generated in insect cells is conformationally distinct from the native TPO, is insoluble and enzymatically inactive, consistent with the difficulties associated with its purification and crystallisation.     

Regulatory perspectives from Japan - comparability of biopharmaceuticals.

In Japan there is no official guideline about comparability assessment of biotechnological products at present. However, there is some notifications which should be referred to, when the manufacturer changes the manufacturing process. Here, regulatory perspectives from Japan on the comparability assessment are presented. When establishing the comparability of biotechnological products derived from different manufacturing processes and the validity of modified manufacturing process, rational step-by-step approaches based on both product and process aspects would be useful. At first, relevant physicochemical and biological properties of products including purity, impurity profiles and stability should be compared before and after the manufacturing change, depending on the type and nature of the desired products. It is also necessary to examine the capacities of the new manufacturing process for ensuring the consistent production of the active protein product as well as the anticipated elimination of potential impurities and contaminants. Further relevant assessment of preclinical and clinical comparability of product may be necessary in some cases.     

In vitro and in vivo modifications of recombinant and human IgG antibodies

Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules.     

In vitro and in vivo modifications of recombinant and human IgG antibodies

Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules.     

Comparability studies of new 3rd generation recombinant human factor VIII GreenGene F after improvement of formulation and viral inactivation/removal process

A new 3rd generation recombinant factor VIII (rFVIII), GreenGene F (WHO INN: beroctocog alfa), which is a highly homogenous B-domain deleted FVIII protein comprising of two peptides as heavy chain (A1 and A2 domain) and light chain (A3, C1, and C2 domain) at 80 and 90 kDa, was developed from its predecessor product GreenGene (2nd generation product previously approved by Korea FDA after clinical studies in South Korea) by process improvements of i) addition of Solvent/Detergent treatment for virus inactivation, ii) nanofiltration (20 nm pore size) for viral removal and iii) alterations to an albumin-free formulation to minimize the risk of viral contamination. An assessment of comparability between the two products was made to see if process improvements for safer product manufacturing affected the rFVIII structural and functional characteristics. Physicochemical and physiological characteristics were observed, in vivo efficacy following a single intravenous administration to FVIII knock-out mice and toxicity by various GLP in vivo tests were evaluated. All results showed equivalence, proving that no changes in protein characteristics of rFVIII occurred from process changes in formulation, viral inactivation, and viral removal which minimize the risk of pathogen transmission to enhance safety.