Biosimilar Surge Driving Need for Antibody Characterization
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Accurate protein characterization is particularly important in the biopharmaceutical industry, where product modifications can affect efficacy and safety.
In this two-part series, we speak to Matthew Lauber, a consulting scientist at Waters. Here, Matthew describes the importance and challenges of biotherapeutic protein characterization, and discusses different techniques used for charge variant analysis.
In the second part, Matthew provides insights on how the biopharmaceutical industry benefits from advances in chromatography technology.
Abbreviations: CE: capilliary electrophoresis; cIEF: capillary isoelectric focusing; IEX: ion-exchange chromatography; LC: liquid chromatography; mAbs: monoclonal antibodies; QC: quality control
Michele Wilson (MW): Why are reproducible and accurate analytical methods so important for protein characterization?
Matthew Lauber (ML): Many biotherapeutics have not been universally available to many demographics due to high costs, yet they continue to be hugely impactful drug products. At the same time, we have seen a surge in the development of biosimilars (generic forms of a biological drug) due to the expiry of biotherapeutic patents.
Illustrating the bioequivalence of these so-called biosimilars is not as simple as with pharmaceutical drugs as they consist of relatively large, complex proteins that can be challenging to characterize. On top of this, the biopharmaceutical market is burgeoning with from innovator companies pursuing new modalities and mAb-based therapeutics.
Consequently, the advancement in biosimilar development and continued research, development, and commercialization of new biotherapeutic monoclonal antibody (mAb) entities have driven the demand for accurate, reproducible analytical methods for protein characterization.
MW: What makes the manufacture and characterization of mAbs so challenging?
ML: A number of factors make the production and characterization of monoclonal antibodies (mAbs) extremely challenging. Firstly, mAbs are highly complex biological macromolecules, which for decades have provided long-term popular treatments for many life-threatening and chronic diseases such as cancer. They contain numerous domains, and the defined specificity of a mAb depends on the binding region for an antigen, located in the antigen binding fragment region. mAbs may also have size and charge variants, and are susceptible to enzymatic post translational modifications such as glycosylation and disulfide bond formation, which ultimately confer a very unique profile to every mAb. On top of this, mAbs are also susceptible to chemical modifications, such as oxidation and deamidation, based on exposure and aging during purification and storage.
MW: Can you explain the regulatory landscape for biotherapeutic protein characterization?
ML: The regulatory landscape indirectly influences the techniques employed for biotherapeutic protein characterization. Regulatory bodies such as the U.S. Food and Drug Administration and the International Conference on Harmonization (ICH) work with the biopharmaceutical industry on new drug applications and sometimes provide guidance on characterization techniques including physiochemical characterization, biological characterization, immunochemical characterization, and impurities and contaminants analysis. The methods recommended for each of these can vary, but there is a requirement for molecules to be well characterized.
MW: Which analytical techniques are preferred for the characterization of mAb primary structure?
ML: Charge variant analysis is a regulatory requirement for biotherapeutic proteins, as stated by ICH and WHO (World Health Organization) guidelines, which also give the recommended standards for the evaluation of biologics.1-3 The guidelines recommend the physiochemical characterization of mAb primary structure and the use of liquid chromatography (LC), including size-exclusion chromatography, reverse-phase chromatography, ion-exchange chromatography (IEX), and affinity chromatography. Alternative technologies, such as capillary electrophoresis (CE) and capillary isoelectric focusing (cIEF), are also proposed.
MW: Can you explain charge variant analysis and its role in biotherapeutic protein characterization?
ML: Due to the fact that they are regularly defined as critical quality attributes during biopharmaceutical development, charge variants necessitate careful characterization to guarantee safety and efficacy.4 Charge variants can be present as both acidic (lower isoelectric point species) or basic variants (higher isoelectric point species), depending on the specific manufacturing conditions or the innate properties of a mAb.
Additional charge heterogeneity can be incurred during mAb process development and formulation. As a result, charge variant analysis is employed across the biopharmaceutical pipeline, from the discovery stage through to manufacturing and quality control (QC) phases. Characterizing charge variation early on in the biosimilar development process can mitigate the risk of a drug failing late in the pipeline, as it helps to identify any differences between a biosimilar and innovator drug and it provides information on issues with chemical stability and their physicochemical consequences.
MW: What is the most popular technique for charge variant analysis?
MW: What makes the manufacture and characterization of mAbs so challenging?
ML: A number of factors make the production and characterization of monoclonal antibodies (mAbs) extremely challenging. Firstly, mAbs are highly complex biological macromolecules, which for decades have provided long-term popular treatments for many life-threatening and chronic diseases such as cancer. They contain numerous domains, and the defined specificity of a mAb depends on the binding region for an antigen, located in the antigen binding fragment region. mAbs may also have size and charge variants, and are susceptible to enzymatic post translational modifications such as glycosylation and disulfide bond formation, which ultimately confer a very unique profile to every mAb. On top of this, mAbs are also susceptible to chemical modifications, such as oxidation and deamidation, based on exposure and aging during purification and storage.
MW: Can you explain the regulatory landscape for biotherapeutic protein characterization?
ML: The regulatory landscape indirectly influences the techniques employed for biotherapeutic protein characterization. Regulatory bodies such as the U.S. Food and Drug Administration and the International Conference on Harmonization (ICH) work with the biopharmaceutical industry on new drug applications and sometimes provide guidance on characterization techniques including physiochemical characterization, biological characterization, immunochemical characterization, and impurities and contaminants analysis. The methods recommended for each of these can vary, but there is a requirement for molecules to be well characterized.
MW: Which analytical techniques are preferred for the characterization of mAb primary structure?
ML: Charge variant analysis is a regulatory requirement for biotherapeutic proteins, as stated by ICH and WHO (World Health Organization) guidelines, which also give the recommended standards for the evaluation of biologics.1-3 The guidelines recommend the physiochemical characterization of mAb primary structure and the use of liquid chromatography (LC), including size-exclusion chromatography, reverse-phase chromatography, ion-exchange chromatography (IEX), and affinity chromatography. Alternative technologies, such as capillary electrophoresis (CE) and capillary isoelectric focusing (cIEF), are also proposed.
MW: Can you explain charge variant analysis and its role in biotherapeutic protein characterization?
ML: Due to the fact that they are regularly defined as critical quality attributes during biopharmaceutical development, charge variants necessitate careful characterization to guarantee safety and efficacy.4 Charge variants can be present as both acidic (lower isoelectric point species) or basic variants (higher isoelectric point species), depending on the specific manufacturing conditions or the innate properties of a mAb.
Additional charge heterogeneity can be incurred during mAb process development and formulation. As a result, charge variant analysis is employed across the biopharmaceutical pipeline, from the discovery stage through to manufacturing and quality control (QC) phases. Characterizing charge variation early on in the biosimilar development process can mitigate the risk of a drug failing late in the pipeline, as it helps to identify any differences between a biosimilar and innovator drug and it provides information on issues with chemical stability and their physicochemical consequences.
MW: What is the most popular technique for charge variant analysis?
ML: LC offers a robust, high resolution approach to biopharmaceutical analysis and, as such, is favored by many laboratories. Its high degree of selectivity assists in the analysis of mAb variants which may have almost identical physicochemical properties. Among potential LC techniques, it is IEX that is widely adopted for the separation and characterization of mAb charge variant species. We have seen great advancements in IEX technology. It is a frequently used modality of LC that separates molecules based on the ionic groups that are exposed on a protein’s surface.
Anion exchangers bind negatively charged entities, while cation exchangers bind positively charged entities. The latter have proven to be very useful for mAbs given that most mAbs tend to bear a positive net charge across a broad range of pH conditions (e.g. pH 6 to 8). In practice, a separation with an ion exchange LC column is also relatively easy to produce. Samples can be adsorbed to the stationary phase under low ionic strength conditions and eluted with a gradient of increasing salt or a change in mobile phase pH.
MW: What are the advantages of IEX chromatography over other techniques?
ML: CE and cIEF have the fundamental benefits of speed of analysis and that they generally require only minimal method development. However, IEX chromatography has a key advantage over CE/cIEF, which is the ability to collect separated species for additional analyses that may include structure-function characterization and tests to confirm biological significance. For instance, an IEX fraction can be collected and used in a ligand binding/surface plasmon resonance study to determine if binding to the therapeutic target is preserved.
By contrast, it has remained impractical to collect satisfactory amounts of CE or cIEF separated species. In addition, recent reports suggest that IEX chromatography can be easily coupled to mass spectrometers for online mass spectrometry (MS) detection,5 or used in multidimensional separations for deep characterization of a mAb.6 This is not as easily facilitated with CE or with cIEF that uses MS incompatible ampholytes. Many analysts therefore consider IEX as being the “gold standard” for charge variant analysis.
MW: Which types of gradients produce the best results and why?
ML: As described earlier, IEX chromatography elution can be achieved using either salt- or pH-based gradients. Both changes in mobile phase composition can be used to disrupt the electrostatic interactions between the protein and the stationary phase. Having a reproducible, flexible, precise IEX method for charge variant analysis is imperative for the biopharmaceutical industry. Although salt-based gradients have traditionally been the obvious approach, a drive to quicken the turnaround of results has led to the consideration of platform pH gradient methods that do not require the tailoring of unique method conditions for each individual target protein. In the past ten years, the popularity of pH-based gradients has grown significantly, primarily for their global applicability to many different mAbs.7
Even though the two gradient modes produce an elution order of mAb variants that is often comparable, pH gradients offer a quicker means to obtaining a high-resolution profile. The reproducibility of pH-based gradients and the ease of their implementation also makes them appealing for method transfer to QA/QC and contract research organizations.
Matthew Lauber was speaking to Michele Wilson, Science Writer for Technology Networks
References:
ML: As described earlier, IEX chromatography elution can be achieved using either salt- or pH-based gradients. Both changes in mobile phase composition can be used to disrupt the electrostatic interactions between the protein and the stationary phase. Having a reproducible, flexible, precise IEX method for charge variant analysis is imperative for the biopharmaceutical industry. Although salt-based gradients have traditionally been the obvious approach, a drive to quicken the turnaround of results has led to the consideration of platform pH gradient methods that do not require the tailoring of unique method conditions for each individual target protein. In the past ten years, the popularity of pH-based gradients has grown significantly, primarily for their global applicability to many different mAbs.7
Even though the two gradient modes produce an elution order of mAb variants that is often comparable, pH gradients offer a quicker means to obtaining a high-resolution profile. The reproducibility of pH-based gradients and the ease of their implementation also makes them appealing for method transfer to QA/QC and contract research organizations.
Matthew Lauber was speaking to Michele Wilson, Science Writer for Technology Networks
References:
- International Conference on Harmonization (ICH) Q6B, Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products, March 1999.
- Guidelines on evaluation of similar biotherapeutic products (SBPs), Expert Committee on Biological Standardization, World Health Organization (WHO), October 2009.
- Guidelines on evaluation of monoclonal antibodies as similar biotherapeutic products (SBPs), Expert Committee on Biological Standardization, World Health Organization (WHO), 2017.
- Singh SK, Narula G and Rathore AS (2016) Should charge variants of monoclonal antibody therapeutics be considered critical quality attributes? Electrophoresis, 37, pp. 2338-2346. DOI 10.1002/elps.201600078.
- Leblanc, Y.; Ramon, C.; Bihoreau, N.; Chevreux, G., Charge variants characterization of a monoclonal antibody by ion exchange chromatography coupled on-line to native mass spectrometry: Case study after a long-term storage at +5 degrees C. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 2017, 1048, 130-139.
- Gstottner, C.; Klemm, D.; Haberger, M.; Bathke, A.; Wegele, H.; Bell, C.; Kopf, R., Fast and Automated Characterization of Antibody Variants with 4D HPLC/MS. Analytical chemistry 2018, 90(3), 2119-2125.
- D’Silva K, Cook K and Cubbon S (2017) Advances in Chromatography for Charge Variant Profiling of Biopharmaceuticals, The Column, 13(9), pp. 28-32.
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