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Confounding signals pose challenges to analytical methods necessary for managing residual impurity removal in biotherapeutic manufacturing.
Managing residual impurities in downstream processing is a challenge, even for traditional biologics such as monoclonal antibodies (mAbs). When it comes to managing residual impurities in downstream processing for emerging biotherapeutics, such as gene therapy or even cell therapy, those challenges are heightened due to the complex nature of the end product itself as well as the complicated bioprocesses needed to manufacture these types of products.
One such challenge in downstream bioprocessing is the requirement that the purification process be reliable and predictable to make product that is suitable for human use. It is thus imperative that during the downstream purification process, impurities such as host cell protein (HCP), DNA, adventitious and endogenous viruses, endotoxins, aggregates, and other species are removed while maintaining an acceptable product yield (1). Furthermore, impurities are also sometimes introduced during the purification process itself, and these must also be removed. Examples of impurities resulting from the processes themselves include leached Protein A, extractables from resins and filters, process buffers, and agents (e.g., detergents) that may have been used for viral clearance (1).
Recovery of an antibody product from mammalian cell culture typically starts with the cell harvest stage, where cells and cell debris are removed. The resulting clarified, filtered fluid is further purified in chromatography, which is a well-established process. In contrast, more complex emerging therapies, such as gene therapy products, present a unique set of challenges for controlling residual impurities. Because current gene therapy products are viral vector-based, this poses additional challenges to controlling the presence of HCPs. For gene therapies intended for severe indications that are placed on an accelerated development pathway, one critical challenge is the conflict between time-to-market pressures and the timelines required to develop a product-specific immunoassay (2).
As with mAbs, HCP content in viral-vector products is a critical quality attribute. HCP content is highly relevant to the safety of a gene therapy product (2). HCPs pose a risk directly through toxicity, hypersensitivity, endotoxin shock, or their own inherent biological activity. They may also pose a risk through immunogenicity, in which the HCPs themselves become the direct targets of the immune response. The presence of residual HCPs can also contain enzymes that can cause a gradual breakdown in protein components, thus compromising product stability (3).
Gene therapies that use viral vectors, such as adeno-associated virus (AAV) vectors, are challenging specifically because they contain viral material. Thus, removing impurities becomes especially complex when trying to distinguish between product and product-related impurities that closely resemble the vector itself. Thus there is a need to optimize both the upstream cell culture processes to reduce biosynthetic generation of these impurities and the downstream purification processes to reduce or remove these impurities without removing the desired product (4).
Appropriate and discerning analytical methods are key to managing residual impurities in emerging biotherapies. The ability to detect specific impurities in viral-vector-based gene therapies, for instance, allows for appropriate removal and process control over process-related impurities. However, the different analytical techniques available to the industry require a range of expertise in not only how to use them, but how to correctly interpret the data generated from these techniques.
The broad range of impurities that can show up in viral vector drug substance/drug product manufacturing needs comprehensive analysis. These analytical techniques include enzyme-linked immunosorbent assay (with specific antibodies), quantitative polymerase chain reaction (PCR) or digital-droplet PCR (ddPCR) assays using specific targeted amplicons, sodium dodecyl sulphate–polyacrylamide gel electrophoresisand capillary electrophoresis, high-performance liquid chromatography, analytical ultracentrifugation, fluorescence-activated cell sorting, and mass spectrometry (5).
Viral vectors themselves can be characterized with analytical techniques similarly used for traditional biotherapeutics; however, because understanding of viruses and virus functions remains incomplete, this fundamental lack of information complicates the issue of thoroughly characterizing viral vectors with certain analytical methods. Viral vectors consist of both protein and nucleic acid components. Methods must be used to characterize not only the viral capsid itself, but also the encapsulated genome (i.e., the “gene therapy”) (5). Complicating that is the challenge of then identifying and separating the product’s genetic signature from the genetic signature of residual impurities at the end of a manufacturing run, particularly impurities that closely resemble the product itself.
Implementing a risk-based strategy for testing residual impurities has the potential to significantly reduce the amount of testing needed while meeting regulatory requirements and ensuring patient safety. To do so, however, requires a full understanding of the biomanufacturing process used for the desired biotherapeutic product as well as appropriate control strategies that minimize impurities at the end of the production process. While building a knowledge base on residual impurities requires investment, that investment pays off in the long term because it enables risk-based residual impurity control and minimizes the need for residual impurity testing (6).
Residual impurities from bioprocessing can be derived from the host cell, the product itself, media components, surfactants, virus inactivation agents, and inorganic or organic contaminants from fixed and single-use process equipment. In addition, the pharmaceutical formulation or a potential breach in sterility can also introduce impurities. Risk-based strategies for controlling these impurities are largely driven by the quantity and heterogeneity of the impurities (6).
Meanwhile, analytical methods that have been previously developed and validated for traditional mAb biologics have also been found to be useful and applicable to detecting the presence of process-related impurities in the AAV vector manufacturing process, including residual HCPs and nuclease-sensitive nucleic acids. However, new analytical methods have been required to characterize and quantify these vector product-related impurities. Some newer methods need to be validated to the standards required for all licensed biotherapeutic products, however, and the industry still needs further develop analytical methods for AAV-vector-based gene therapy production (4).
Viral vector product-related impurities have been shown to be structurally related to, but distinct and not comparable to, the desired vector product in terms of efficacy and safety. These product-related impurities can include biosynthetic intermediates as well as particles of incorrect composition—such as nuclease-resistant nucleic acid impurities that are packaged in AAV capsid particles. Degraded, oxidized, and aggregated forms of the vector product can also be present as product-related impurities (4).
The overall trend is a rise in both the total number of biotherapuetics being manufactured and also their sophistication and complexity. Fine tuning targeting the signal from the noise in production, the desirable DNA from the impurity, is an ongoing process where new analytic tools are being developed and deployed with increasing success.
1. H.F. Liu, et al., MAbs 2 (5) 480–499 (2010).
2. O. Stamm, et. al., “Detecting Residual HCPs Demands a Holistic Approach,” BioPharm International Biopharmaceutical Analysis eBook (July 2021).
3. S. Pengelley, “Increasing the Depth and Reliability of HCP Analysis Using TIMS-MS,” BioPharm International Biopharmaceutical Analysis eBook (July 2021).
4. J.F. Wright, Biomedicines 2 (1) 80–97 (2014).
5. R. Snyder, “Analytical Methodologies Utilized in Therapeutic Viral Vector Manufacturing,” Pharma’s Almanac, Oct. 2, 2018.
6. C. Challener, BioPharm International 33 (7) 22–25 (2020).
Feliza Mirasol is the science editor for BioPharm International.
Vol. 34, No. 12
When referring to this article, please cite it as F. Mirasol, “Managing Residual Impurities in Complex Biotherapeutics,” BioPharm International 34 (12) 24–25 (2021).