Host Cell Protein Measurement and Control. Stocktrek ImagesGetty Images. Host cell proteins HCPs constitute a major part of process related impurities during biologics production. The amount of residual HCPs in drug product is generally considered a critical quality attribute CQA, due to their potential to affect product safety and efficacy. Therefore, it is a regulatory requirement to monitor the removal of HCPs in drug product during bioprocess development. HCPs are proteins produced or encoded by the host organisms used to produce recombinant therapeutic proteins 1. Recombinant therapeutic proteins are usually produced by genetically modified prokaryotic or eukaryotic host cells using cell culturefermentation technology 2. Genetic engineering allows the host cells to be transformed to produce a protein of interest selectively. During the recombinant protein production, host cells also coproduce proteins related to the normal cell functions such as cell growth, proliferation, survival, gene transcription, protein synthesis, and etc. Other non essential proteins may also be released to the cell culturefermentation as a result of cell apoptosisdeathlysis. H. Kaji, G. CamciUnal, R. Langer, A. Khademhosseini Engineering systems for the generation of patterned cocultures for controlling cellcell interactions. Figure 1. CRISPRdCas9Based Applications to Study and Manipulate the Epigenome. Main applications of the CRISPRdCas9 system to study and manipulate the epigenome. Antibody Engineering Volume 1 Pdf' title='Antibody Engineering Volume 1 Pdf' />In general, apart from the therapeutic protein of interest, all endogenous proteins co expressed by the host cells are called host cell proteins 2. Risks Associated with HCPs. HCPs constitute a major group of process related impurities in a drug product. The risks associated with HCPs are primarily immunogenicity. HCPs are complex mixtures with diverse physiochemical and immunological properties 2. National Plumbing Code Of Canada there. Almost all HCPs carry clinical safety risks as foreign proteins due to the potential to elicit immune response in humans. In addition, some HCPs can also act as adjuvants to enhance immune response to a drug product 1, 3. Certain HCPs with proteolytic activity can also affect drug product stability and efficacy if not adequately removed or inactivated 4. HCPs have the potential to affect both the safety and efficacy aspects of a given drug product. Antibody Engineering Volume 1 Pdf' title='Antibody Engineering Volume 1 Pdf' />Molecules, Volume 21, Issue 8 August 2016 Issues are regarded as officially published after their release is announced to the table of contents alert mailing. In a typical recovery process for a therapeutic monoclonal antibody, the highest concentration of HCP is detected in the harvested cell culture fluid HCCF, and then. The risks associated with HCPs are often assessed by a combination of downstream process capabilities, residual HCPs levels, the maximum dose, route of administration, dosing frequency, toxicological data, and clinical data 1. Although it is a common understanding that HCPs pose clinical safety risk due to their potential to elicit an immune response, it is difficult to demonstrate which HCP and in what concentration may cause immunogenicity problems in humans 1. Theoretically, preclinical pharmacological and toxicological evaluations can be performed with the presence of different amounts of HCP impurities however, the evaluation results are mostly irrelevant because the magnitude and nature of the immune response depends on the homology of the amino acid sequence, residual HCPs amount, and product dosing regimen. Digital Anarchy Flickr Crack'>Digital Anarchy Flickr Crack. For this reason, a risk control strategy is applied through the development of robust downstream bioprocess to remove HCPs to as low a level as possible or to undetectable levels in drug substanceproduct 1, 3, 5. Wifi Key Cracker Software. The detectability of residual HCPs, however, also depends on the method of detections sensitivity. Industry addresses this risk by meticulous method development and the use of multiple technologies to evaluate all potential HCPs that might coproduce or copurify with drug product during bioprocess development. Regulatory Requirements for HCPs Measurement and Control. According to International Conference on Harmonization ICH guidelines Q6. B, For host cell proteins, a sensitive assay e. In the case of an immunoassay, a polyclonal antibody p. Ab used in the test is generated by immunization with a preparation of a production cell minus the product coding gene, fusion partners, or other appropriate cell lines Clearance studies, which could include spiking experiments at the laboratory scale, to demonstrate the removal of cell substrate derived impurities such as nucleic acids and host cell proteins may sometimes be used to eliminate the need for establishing acceptance criteria for these impurities 6. FDA expects Whenever possible, contaminants introduced by the recovery and purification process should be below detectable levels using a highly sensitive analytical method 7. The European Medicines Agency EMA guideline CPMPBWP3. In summary, for HCP, whatever the product and production system, residual HCP have to be tested for on a routine basisAs such, it is currently required that HCP be routinely monitored at the purified bulk level, using suitable analytical assays. Results from batch to batch should be consistent and meet specification limits 8. Regulatory agencies from other countries and emerging markets may have their own wording on HCP control, but it is generally accepted that a sensitive, validated method is required to monitor residual HCPs in accordance with ICH guidelines. The allowed amount of residual HCPs in final bulk material is determined on a case by case basis, commonly in the 1 to 1. Measuring and Monitoring Residual HCPs. To date, immunoassay, commonly in the form of sandwich enzyme linked immunosorbent assay ELISA see Figure 1, remains as the industry gold standard for HCP measurement due to its high sensitivity and high throughput 3, 5. The HCP composition and abundance are unique to their respective host and the manufacturing process used for biologics production. Meanwhile, different host cells and manufacturing processes may produce certain HCPs in similar abundance. The number of proteins derived from host cells varies significantly from host to host. For example, Escherichia coli E. Chinese Hamster Ovary CHO cells have 3. Although not every host gene will be transcribed and translated to protein, the complexity of host genome and the post translational modification present in mammalian cells make it almost impossible to understand the complete HCP composition in a given manufacturing process. Because these HCPs are potentially immunogenic, a commonly accepted method to evaluate the presence of HCPs is through an immunoassay. In theory, an HCP mixture injected into an animal, such as a rabbit, goat, or chicken, will elicit an immune response, and the animals will generate anti HCP antibodies against these foreign proteins. Although the identities of all HCPs are not known, the polyclonal antibodies raised in animals should be able to recognize most, if not all, of the proteins contained in the HCP mixture. Using these polyclonal antibodies, a multi analyte sandwich ELISA can be developed see Figure 1. In Figure 1, capture antibodies enrich the HCPs from sample and immobilize them to a 9. Then, detection antibodies, conjugated directly with an enzyme or through a biotin avidin magnification, bind to the captured HCPs. An enzyme, commonly horseradish peroxidase HRP, can catalyze the substrate to generate a colorimetric, chemiluminescent, or fluorescent signal that correlates with the amount of HCPs in the test sample 5. Figure 1 Schematic view of sandwich enzyme linked immunosorbent assay ELISA used for host cell protein HCP detection and measurement. Generic HCP ELISA developed using polyclonal antibodies raised against parental cell lysate or cell culture supernatant allows the detection of a majority of HCP species, but may not be able to detect a subgroup of proteins specific to a certain manufacturing process.