Research on Genetically Engineered Drug Host Cell Proteins

Genetically engineered drugs are introduced into host cells such as Escherichia coli, yeast or mammalian cells through recombinant technology to construct engineering strains or cell strains, and then undergo specific expression, assembly, folding and post-translational modification processes to produce drugs with corresponding Bioactive macromolecules. Endogenous proteins derived from host cells are called host cell proteins (HCPs). Their composition is complex. Depending on the selected host cells and production processes, the isoelectric point (3-11), hydrophobicity, relative the molecular mass (generally 5000~250000) distribution varies significantly. Cellular HCPs in genetically engineered drugs may cause different human reactions when administered, because specific HCPs have potential “adjuvant effects”, generate immune responses against impurities in HCPs, or cause allergic reactions or even anaphylactic shock. In addition to safety issues, the presence of HCPs can also affect product quality. For example, HCPs can trigger aggregation or fragmentation of therapeutic proteins. In addition, it was found that certain HCPs promote the degradation of polysorbates, a type of non-ionic surfactant widely used in the preparation of buffers to stabilize proteins. The degradation of polysorbates will affect the stability of protein drugs. The protease activity in HCPs can also affect the protein composition in the culture supernatant, thereby affecting the subsequent protein purification process or the stability of long-term storage of protein drugs. In addition, HCPs may undergo post-translational modifications, making quantification and characterization more difficult. In view of the fact that cellular HCPs in genetically engineered drugs may cause safety and effectiveness issues, the detection of their removal efficiency and cell limit is an important parameter for drug release and clinical research, and is one of the critical product quality attributes (CQA) of drug quality control. Therefore, although only a few clinical adverse events have so far been attributed to HCPs impurities, in order to ensure product safety, HCPs must be accurately characterized and quantitatively analyzed through highly sensitive analytical methods. Factors Influencing HCPs The quantity and composition of HCPs are affected by the entire production process. Among them, the expression pathway is very important. Depending on the host cells and culture conditions, the types of HCPs vary greatly, ranging from hundreds to thousands. Many host cells such as E. coli, mammalian cells, NSO, SP2/0 and the human embryonic kidney cell line HEK293 have been used for biopharmaceutical production. During mammalian cell culture, recombinant proteins are often secreted from cells into cell culture fluid (CCF), which contains HCPs. In addition, due to the death of some cells, soluble intracellular proteins are released into CCF, and some artificial operations (such as centrifugation, filtration, etc.) can also cause cell lysis. Therefore, harvested CCF usually contains secreted HCPs and intracellular HCPs. When this protein mixture is incubated in a fermenter, other changes in HCPs occur due to enzymatic activity (such as proteases or sialidases). HCPs analysis provides information about the composition of substances entering the downstream process and the removal rate of HCPs at each purification step. In some cases, HCPs can even be combined with or co-purified with certain protein drugs. Process characterization and validation studies are needed to illustrate which process steps can remove HCPs and also to demonstrate the stability of these steps in removing HCPs. Therefore, detection of HCPs is an important part of purification process development and helps ensure production consistency. Finally, stable and highly sensitive detection methods are needed to detect HCPs of drug proteins.