Genetic Engineering & Biotechnology News

SEP15 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Genetic Engineering & Biotechnology News | GENengnews.com | SEPTEMBER 15, 2017 | 13 duce two markers that are commonly inves- tigated using ELISAs —Protein A and host- cell proteins (HCPs). Protein A Protein A is a bacterial protein produced in Staphylococcus aureus that binds to IgGs with affinity. Recombinant Protein A has found wide use in affinity purification of antibodies and in commonly used immu- nodetection and visualization techniques. It is commonly used by the industry to purify monoclonal antibodies (mAbs) from har- vest cell culture supernatant. 1 Protein A affinity chromatography has been shown to be highly selective for mAbs, resulting in a purity greater than 95% start- ing from complex samples (Figure 1). It became the method of choice by achieving high purity and yield in a single unit opera- tion, simplifying manufacturing operations, and reducing times and effort required to develop a purification process. In a typical mAb purification process, the protein-rich supernatant is harvested from hybridoma cells in culture. Then, dur- ing Protein A affinity chromatography, an- tibodies are captured at a neutral pH and eluted at an acidic pH. After the inactivation of adventitious viruses, cation-exchange chromatography is performed to remove HCPs, mAb aggregates, and antibody frag- ments, followed by anion-exchange chro- matography, which removes DNA, leached Protein A, and other contaminants. Follow- ing these filtrations, the biologic is ready for use. 2 However, Protein A is known to participate in a number of biological func- tions, including immunogenic, toxic and/ or mitogenic activities—thus, the quantifi- cation of potential Protein A contaminants from antibody preparations for therapeutic use is necessary. Host-Cell Proteins HCPs are a residual contaminant that can appear in the production of a biologic. They are produced by organisms as a side prod- uct to the intended protein. While there are many different possible host cells from which to choose, many facilities opt to use Esch- erichia coli or Chinese hamster ovary (CHO) cells as model organisms (Figure 2). E. coli has been widely studied and its genome and processes are well understood. It constitutes the easiest, quickest, and most cost-effective method of production of protein. CHO cells are highly efficient, as they can produce re- combinant protein on the scale of 3–10 g/L of culture with less variation if long-term protein production is needed. Although HCPs are necessary for natu- ral cellular functions and some may ac- tually be completely harmless, they are sometimes carried through the purification process and considered undesirable to have in the final product. Every year, consider- able effort is expended in the industry to remove HCPs from biologic mixes. This is complicated by the tendency of HCPs to be present in very small quantities (usually on the ng/mL scale), making it additionally important to quantify HCP residuals in the production of biologics. 3 Many techniques have been developed for HCP analysis, such as gel electrophoresis or Western blot. However, the most commonly used method remains ELISA. Conclusion When pursuing production of a protein, introduction of contaminants is unavoidable. ELISAs are well suited for this problem: they are sensitive and specific, two qualities that allow manufacturing facilities to detect the presence of contaminants. Specifically, ELI- SA technology is typically used to investigate Protein A and HCP contaminants. When us- ing an ELISA, it is of the essence to achieve a verifiably accurate and precise result; as very often, the entire process will depend on the final purity of the biologic. Drug Discovery Tutorial Figure 2. Sample standard curve for a commercially available Chinese hamster ovary (CHO) host-cell protein ELISA kit. Jonathan Weinreich (jweinreich@ enzolifesciences.com) is associate product manager at Enzo Life Sciences. Web: www.enzolifesciences.com. References available online.

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