Achieve Perfection with Better HCP Detection

Biopharmaceuticals, such as monoclonal antibodies and recombinant peptides, are important therapeutics used to treat human diseases. The key features of these innovative medicines are clinical effectiveness, high specificity for their human target, long half-life, target coverage, and low risk for "off-target" side-effects. There are, however, some differences in the overall safety testing paradigms for small molecules and biopharmaceuticals in term of quality and toxicology. These differences are associated with both the manufacturing processes involved and the molecules themselves. Taking the example of biopharmaceuticals, living cells represent the factories where complex molecular entities are synthesized. As a result of this, safety testing for this class of drugs includes adventitious agent testing (e.g. viruses, mycoplasma, transmissible spongiform encephalopathy agents, protein A and host cell protein (HCP) contaminations) not normally needed with small molecules.

Host cells such as E. coli, yeast cells, or mammalian (e.g. Chinese hamster ovary (CHO) cells) are used to express proteins that ultimately become recombinant therapeutic products. Residual DNA and HCPs are impurities that may remain in a recombinant drug product even after purification. The FDA requires manufacturers to report how much residual DNA and HCPs from the host cell remains in the drug substance after purification. Levels of these materials are also monitored during process development and validation, and help manufacturers select the best methods to reduce these impurities. Because recombinant therapies are themselves proteins, detection of residual host cell DNA against the backdrop of proteins is relatively more straightforward than the detection of residual HCP. In contrast, many different types of proteins are expressed by a host cell. Thus, residual HCP measurement methods must be able to sensitively identify multiple proteins in the presence of other high-abundance proteins and drug product (often 1-100ng HCP per mg of drug product or ppm), which can, unfortunately, be quite challenging.

Host cell proteins occur over a wide range of concentrations and they can even interact directly in a non-covalent manner with the expressed recombinant proteins, notably with monoclonal antibodies (mAbs). The consequences can be three-fold: the patients receiving treatment with a mAb might develop an immune response against HCPs; the efficiency of the mAb in binding its target epitope might be diminished by blockade of the binding site; and the overall stability of the biopharmaceutical might also be affected with the potential binding of proteases and other reductases. In a research article published in Biotechnology Progress, Dr. Aboulaich and colleagues from MedImmune developed an approach to identify which specific HCPs bind to mAbs. Four purified target mAbs were immobilized on separate sepharose resins via cross-linking and were incubated with host cell proteins isolated from the culture supernatant of non-producing CHO cells. The HCPs capable of binding mAbs along with the mAbs were recovered and identified by tandem mass spectrometry. A similar distribution of 36 intracellular proteins were found to bind all four mAbs, which represent 70-80% of mAb-bound HCPs. Some of the identified HCPs, namely cathepsin B, MMP-19 or protein disulfide isomerase (PDI) can affect dramatically the structural integrity of mAbs. The designed approach was then employed to test different wash modifiers and determine which one was more apt to dissociate the interactions between the identified HCPs and the mAbs. The dissociation by wash modifiers was proven to be pH-independent and antibody-dependent. Using in-house CHO HCP ELISA kit, they also demonstrated that the presence of tetramethylammonium chloride and arginine in these wash buffers can enhance significantly the clearance of bound HCPs (about 20% of control). Altogether, the authors proved that by combining immobilization of a mAb onto a column, testing of different wash buffers, and quantification of HCPs by ELISA, a truly purified antibody with retained efficacy can be obtained with the development and optimization of an antibody-specific purification protocol.

Enzo Life Sciences offers a comprehensive product portfolio to identify protein impurities in biopharmaceuticals including HCPs and protein A, some of which are described below:

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