Biologic monoclonal antibody (mAb) drugs are not just more targeted than small-molecule drugs, they are also larger, more complicated, and more variable. Consequently, biologic mAb drugs, which generate sizable revenues, may enjoy some degree of protection from biosimilars, follow-on biologics that are similar but not identical to their corresponding reference biologics. To obtain the maximum degree of protection, biologic mAb drugs need to be intensively and comprehensively characterized. Differences between mAbs and biosimilars, it may be argued, are not merely incidental, but crucial to biological activity and therapeutic action.
Biologic/biosimilar differentiation is just one reason to enhance mAb characterization. Other reasons—potentially more compelling reasons—include the need to collect metrics that can be used to screen mAb drug candidates, optimize production processes, and satisfy increasingly stringent regulations. Also, as mAb processes become more sophisticated, increasingly subtle mAb characteristics become significant.
Back when the U.S. Food and Drug Administration approved the first mAb drug, muromonab-CD3 (Orthoclone OKT3), making mAbs was a complex and cutting-edge undertaking. It involved fusing cells—in the case of Orthoclone OKT3, one from a mouse’s spleen and one from a tumor—to create an expression cell line followed by multiple harvesting steps. Yields were low, and production was costly.
Today, mAb production is almost routine. Cell line development and manufacturing techniques have advanced, and more efficient production and harvesting platforms have been developed. Changes such as these mean that the mAb industry faces more nuanced challenges in elucidating mAb molecular structures and characteristics.
Fortunately, industry can meet these challenges by deploying advanced characterization technologies. Such technologies can, for example, helping industry find mAb candidates faster, says Anis H. Khimani, PhD, head of strategy and marketing at PerkinElmer. “Monoclonal antibody characterization is important for the biopharmaceutical industry,” he says, “because it enables selection of the therapeutic molecule representing greater specificity, efficacy, stability, and functionality.”
Characterization through development and production
The rationale for characterizing a mAb differs at each stage of the development and production process. Understanding what you are looking for and why is critical. “It is important,” says Gunnar Malmquist, PhD, principal scientist at GE Healthcare, “to distinguish between the mAb characterization that takes place during drug development and the mAb characterization that is performed during process development and routine manufacturing.” The former is used to determine that you have the correct mAb and to establish potential critical quality attributes of the antibody. The latter depends, to some extent, on these attributes. “They will identify which product-related impurities you will need to consider during process development and subsequently during manufacturing,” Malmquist advises.
The analysis methods used during process development and routine manufacturing fall into three different categories: process control and lot release; stability indicators; and characterization, which is used to confirm that drug properties remain consistent and to confirm acceptable levels of product-related impurities.
Properly executed, analytical methods help manufacturers detect high-molecular-weight aggregates resulting from the mAbs sticking together during production. Likewise, analytical techniques are used to identify other impurities like host cell proteins or leached Protein A.
To illustrate the importance of such steps, Maryann Shen, PhD, LCMS global marketing program manager, Agilent Technologies, discusses how they relate to a representative characterization activity, glycan profiling. “Many approved biopharmaceuticals are glycoproteins,” she says. “Glycans can play an important role in drug efficacy and safety. Regulatory agencies demand detailed analysis of glycosylation. Glycan profiling is a very common step in the characterization of the mAb.”
Characterization challenges posed by heterogeneity
Biomanufacturing always involves variability. While production advances have brought greater consistency, the use of living cells in the process makes it impossible for all variables to be controlled.
For mAbs, this variability usually impacts the product’s quality attributes more often than the molecule itself, suggests Michael Walker, PhD, technical expert, LC-MS protein analysis, Intertek Pharmaceutical Services. “This route of manufacture,” he points out, “leads to heterogenicity in production lots, specifically in relation to differences in post-translational modifications, which are hugely significant as they can impact both the efficacy and safety of the final product.
“Understanding the structural characteristics of the heterogeneous population is consequently not so much concerned with ensuring the correct mAb is formed, although identity testing would always be included, but [with] developing safe, effective therapies through identification of the critical quality attributes. Ideally, these attributes, which need to be controlled through production, should be continuously monitored to ensure the continued efficacy and safety of the product.”
This assessment of modifications is an area of active research for industry says Malmquist. He told us, “The extent of post-translation modifications lead to a severe variability of the mAb structure and therefore to a large number of potential product related impurities that need to be characterized.
“An emerging trend [to achieve this] is to look at so-called multiattribute methods, which are based on a combination of peptide mapping and mass spectroscopy. The goal is to assess multiple quality attributes with one analytical method.”
Characterization techniques common to the lab and the factory floor
Another characterization trend has seen drug companies take analytical techniques usually carried out in the laboratory into the production space. According to Malmquist, the approach, which involves firms placing analytical instrument near manufacturing lines, “will increase data frequency, decrease response times, and improve process control.”
Eventually, real time release testing may even be possible which, Malmquist says, would significantly reduce release time by ensuring quality targets are met during the manufacturing process.
Comparing reference biologics and follow-on biosimilars
Biopharmaceuticals, as stated above, are less likely to face competition than small-molecule drugs. Primarily this is due to variability inherent in production, which makes it hard for a company to replicate another’s product. That said, off-patent biopharmaceuticals can still face competition from biosimilars. Although biosimilars are not generics, they are conceptually similar.
Biosimilar requirements vary from market to market. In general, securing a biosimilar’s approval is a matter of showing that the molecule’s active properties are similar to those of a reference product that has already been cleared by regulators. To date, many of the approved biosimilars are versions of mAb-based therapies.
“The biosimilar manufacturer,” Malmquist says, “will have to exert a substantial and increased characterization effort in demonstrating structural similarity to the originator profile to benefit from the simplified regulatory framework available.”
The biopharma industry also compares biologics and biosimilars for competitive reasons. It is now common for firms to try to develop as complete and detailed a description of their product’s molecular properties as possible to make it harder for biosimilar firms to create matching drugs.
Incorporating advanced analytical technologies
Technology advances support more detailed analyses, which are, Walker points out, becoming increasingly routine as biomanufacturers respond to regulatory demands. “Data-rich techniques like mass spectrometry allow more critical quality attributes to be monitored in a single assay to improve process development,” he continues. “The increased throughput and data integrity that technological improvements have allowed open up new parts of the manufacturing pipeline for complex analytical techniques.”
The emergence of next-generation mAbs, antibody drug conjugates (ADCs), and fragment-based drugs is also impacting how industry uses characterization technologies.
“Beyond conventional mAbs, related products such as Fc fusion proteins or bispecific antibodies (bsAbs) and antibody-fusion proteins are in development,” Walker details. “Each of these brings specific characterization challenges that may require new approaches and technologies.
“For example, Fc fusion proteins are susceptible to proteolytic cleavage. They also have the potential to form higher levels of high-molecular-weight aggregates as compared to conventional mAbs. This drives the need for a definitive suite of approaches to monitor stability and occurrence of aggregation.
“If bispecific antibodies introduce a new product-related impurity, the potential for mismatching of protein subunits needs to be controlled. This is analytically challenging as often there is a high degree of conservation between chains. These types of molecules have led to an increased interest in native mass spectrometry methods hyphenated to methods such as size-exclusion chromatography and capillary electrophoresis.”
Consolidating characterization chores
Another dynamic impacting biopharma’s approach to mAb characterization in the drive for efficiency. Every company wants to get product to market as quickly as possible. In the mAb space, the focus is on completing the necessary analysis steps—including characterization—as accurately and efficiently as possible.
“Currently, there is a high desire to combine multiple tasks into one single method, as researchers require both detailed information and faster results,” explains Shen. “Monoclonal antibody characterization is complicated, and many tasks are involved in the process. These usually require labor-intensive sample preparations and time-consuming analyses. It is desirable to have instruments that provide reproducibility, robustness, and ease of use.”
Walker has also observed this trend: “Monoclonal antibody characterization involves a diverse set of advanced analytical techniques, many of which require specialist equipment and training. As some technologies are maturing, there has been a move toward more automated sample preparation and data analysis to reduce timelines.
“An example of this is with mass spectrometry, where hardware and software are becoming more user-friendly, reducing the time taken by specialist operators. There is still, however, some work to be done in this area so that relatively inexperienced operators can access the technology.”
A productive dialectic: characterization needs and technological capabilities
Biopharmaceutical industry demand for more detailed mAb characterization systems will continue to be a major development driver. “The analytical field has witnessed continued development of technologies to enhance characterization, isolation, and purification of mAbs,” states Khimani. “The chromatographic and electrophoretic techniques have been combined with mass spectrometry with significant improvements to the sample preparation requirements.”
Khimani cites automated capillary electrophoresis–based separation technologies and advances in assay development as examples of the work being done, explaining that they “have enabled evaluation of mAb in native conformation.”
“With continued development of next-generation technologies and tools, characterization of mAb will continue to evolve,” he predicts. “It will be a growing space for investigators within the biopharma and biologics segments.”