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USP offers strategies to minimize residual impurities in downstream processing.
Myriad choices confront manufacturers before they even consider optimizing downstream processes. Each decision directly influences what final options will be available on arriving at the final stages of downstream processing and then interlock with fill/finish steps, which demand still more evaluation and forethought. Biopharmaceutical expression systems range from yeast and insects to mammals and plants. Bioreactors to house these systems might be stirred-tank, air-lift, fixed-bed, hollow-fiber perfusion, or suspension, perhaps even hybrid combinations thereof. All of these lead to a gamut of membrane separation, centrifugation, extraction, precipitation, and chromatography technologies. Biology and financial costs then ultimately dictate what works best for each company’s, institute’s, or organization’s reach and ambition.
A group from McMaster University set out on experiments they hoped would benefit manufacturers of therapeutic viruses who are experiencing issues with losses during sterile filtration, by demonstrating that improving host cell protein removal in earlier purification steps can enhance sterile filtration performance. That team observed that, “Viruses may adsorb directly to the membrane through a combination of electrostatic and hydrophobic effects; this is influenced by membrane chemistry, the pH and ionic strength of the solution, and the presence of any additives or other components in the solution. For biopharmaceutical applications, virus formulation buffers are often precisely optimized in order to maximize virus stability, which leaves little room for modifications aimed at improving filtration performance” (1). This was the main goal of their study.
They went on to emphasize that, “One modification that can be made to improve the filtration process is to minimize the amount of residual protein and DNA impurities … Particular concerns relating to product safety include the oncogenicity of residual DNA and the immunogenicity of residual proteins. Although impurities are typically removed during downstream processing prior to sterile filtration, some small residual amounts can remain. This can be problematic, as small residual amounts of DNA have been shown to mediate aggregation in adenovirus and lead to reduced recovery (ratio of infectious virus in the filtrate to infectious virus in the feed) after sterile filtration” (1). The McMaster description of this problem sets the scene for why minimizing impurities is of fundamental importance. In the following interview, Fouad Atouf, senior vice president, Global Biologics, US Pharmacopeia (USP), discusses how best to mitigate and minimize residual impurities.
BioPharm: Can you contrast therapies using Chinese hamster ovary (CHO) cells versus more complex human cell lines, and a need to develop suitable immunoassays?
Atouf (USP): Impurities derived from production host cells such as residual HCP [host cell proteins] and HCD [host cell DNA], need to be addressed regardless of cell line. We know more about CHO because of volume of usage for production of mAbs [monoclonal antibodies]. The principle for testing HCPs or HCDs will be the same in other cell lines; acceptable levels will evolve as we learn more about products using cell lines (other than CHO) to produce viral vectors in cell and gene therapy (CGT) applications. A couple of points to highlight regarding other types of impurities:
There are many unique assays for cell-based products: transduction efficiency, LV integration, off-target editing, empty capsids.
BioPharm: High-risk HCPs can be highly immunogenic. Can you mention a few main ones to watch for and how best to mitigate against?
Atouf (USP): Many cell lines are used to produce viral vectors (AAV [adeno-associated viruses], LV [lentivirus] for CGT), and there is a risk for HCPs carried to viral vectors used for GT. Not much has been reported on HCPs derived from HEK293, Sf9 cells—for example—and I anticipate that as these other cell lines gain high-volume usage (just like CHO), we will begin to learn more about the type of biological impurities, whether these are HCP or HCDs. Beyond HCPs, and regarding gene therapy products, immunogenicity concerns can be linked to from AAV capsid composition, and expressed transgenes is a bigger concern.
BioPharm: How can new understanding(s) be integrated into monographs and guidance documents to galvanize improvements?
Atouf (USP): As new impurities are identified and technologies are deployed to identify/quantify these impurities, the methods and relevant reference standards will be introduced into pharmacopeia standards. It starts with stakeholders’ engagement through workshops and roundtables, etc. before initiating collaborative studies for evaluation of the methods and standards. USP now uses iterative and stepwise approaches for standards development. For example, we have been introducing analytical reference materials to support measurement of high-risk HCPs in products made in CHO cell lines. Pharmacopeial standards are revised to support adoption of new technologies and new production systems. For example, USP–NF general chapter <509> Residual DNA Testing supports measurement of residual DNA in products made in CHO and E. Coli. For CGT and vaccines applications, other cell lines are used and methods and standards to support this type of measurement will be introduced.
BioPharm: Are there effective or novel methods to manage residual impurities in downstream processing, especially for those distinguishable from the desired product only by genetic signature?
Atouf (USP): For HCPs impurities for bioproduction cell lines, the big thing that is happening for [the] past few years is the shift to use mass spec for detection and identification as an orthogonal approach to immunoassays. Again, much of the work is on HCPs from CHO, and we don’t know what we don’t know about other cell lines that are getting more usage in manufacturing. An example of trends is the use of NGS [next-generation sequencing] for adventitious agent testing (e.g., mycoplasma) in cell culture. That said, it’s not only adventitious agent testing, but impurities also need to be addressed during process development and product characterization as well. NGS is powerful and versatile, but complex. A lot of development teams don’t have the expertise to use it appropriately or cost-effectively. High performance filtration technologies are great for concentrating impurities into a range where more analytical techniques can be used.
BioPharm: Do you have other observations or comments on this or an adjacent technical area?
Atouf (USP): With the adoption of continuous manufacturing, while the impurities may be the same, the level of these impurities and how you approach testing will need to be optimized. Control of adventitious agents and sterility testing will also need to adjust to new manufacturing technologies; for gene therapies, product-related impurities (full: empty particles or virus particles) [and] process-related impurities (residuals from reagents) will need to be adapted and standardized.
1. Wright, E.; Kawka, K.; Medina, M.; Latulippe D. Evaluation of Host Cell Impurity Effects on the Performance of Sterile Filtration Processes for Therapeutic Viruses, Bioprocessing with Membranes: Filtration and Chromatography. Membranes 2022, 12 (4), 359.
Chris Spivey is the editorial director of BioPharm International.
Volume 36, No. 11
When referring to this article, please cite it as Spivey, C. Paring Down Impurities in Downstream Processing. BioPharm International 2023, 36 (11), 20–21.