Host cell proteins are process-related impurities, expressed by the host cell used for production of biopharmaceutical proteins. During the purification process, the majority of the HCPs are removed, but residual HCP amounts remain in the distributed products, such as monoclonal antibodies, antibody-drug-conjugates, therapeutic proteins, vaccines, and other protein-based biopharmaceuticals. National regulatory authorizations, such as FDA and EMA, require that biopharmaceuticals must be analysed and purified to reduce HCPs to an acceptable level. The HCP acceptance level is evaluated case-by-case and depends on multiple factors including; dose, frequency of drug administration, type of drug and severity of disease. Analysis of HCPs is not simple, since the HCP mixture consists of a large number of protein species, which are unique to the specific host and not related to the intended recombinant protein. The acceptance level of HCPs has commonly been in the range 1-100 ppm due to the detection limit of the established analytical methods. Even with these trace levels of HCPs in the final product reaching the patient, it is unknown if specific residual protein impurities might affect protein stability or immunogenicity in the patient. If the stability is affected, durability of the active substance in a biopharmaceutical could decrease. It is also possible that the effect of the protein might be higher or lower than intended. The degree of immunogenicity on a long-term basis is practically impossible to determine and thus might be a relatively severe threat to the patient’s health.
Safety risk
It is crucial to characterise the HCP population in biopharmaceuticals due to the potential safety risk of introducing foreign proteins into the human immune system. With commonly applied host cell systems such as E. coli, yeast, the mouse myeloma cell line and Chinese Hamster Ovary, the genetic differences between the host system and the human body are many. It is well established that a higher difference to human proteins increases the risk of immunogenicity and thus, a higher level of HCPs is suggested to elicit a more pronounced immune response. Several studies have linked a reduction in HCPs to a decline in specific inflammatory cytokines. Other HCPs may be very similar to a human protein and may induce an immune response with cross reactivity against the human protein or the drug substance protein. The exact consequences of HCPs for the individual patient is uncertain and difficult to determine with the current analytical methods applied in biopharmaceutical approval.
Analysis
During the production process several factors, including the genes of the host cell, the way of product expression and the purification steps, influence the HCP composition and abundance. Several studies report that HCPs often are co-purified along with the product itself by interacting with the recombinant protein. Therefore, the requirements for analytic instruments are extremely high and must be developed further to analyse the entire HCP population more thoroughly in a biopharmaceutical product. Enzyme Linked Immunosorbent Assay is the most commonly applied method for HCP analysis, mainly because it has been the only method with the required sensitivity to detect the low levels of HCPs. Even though the developmental process requires a significant period of work and several research animals, the analysis is rapidly performed and interpreted. However, there are several limitations associated with ELISA for HCP analysis. The HCP quantification relies mainly on the quantity and affinity of anti-HCP antibodies for detection of the HCP antigens. Anti-HCP antibody pools cannot cover the entire HCP population and weakly immunogenic proteins are impossible to detect, since equivalent antibodies are not generated in the process. It is apparent that HCP-ELISAs are insufficient alone for analysis of the HCP population, and therefore orthogonal methods providing complementary information are needed.
For a thorough evaluation on the risk of HCPs in biopharmaceuticals and for proper quality control of the manufacturing, it is of the essence that all HCPs are identified and quantified during the production process and in the final product. A suitable orthogonal method is ideally able to:
Detect varying protein concentrations in a complex sample
Track an ever-changing HCP population and their concentrations during a manufacturing process
Analyse many proteins at once
Measure low abundant HCPs overshadowed by the high abundant target protein product
A method, which fulfills these requirements and emerges as the primary orthogonal method to ELISA, is mass spectrometry. The main advantage of MS is the ability to identify the individual proteins of low abundance, when MS is coupled to liquid chromatography. Recently, the MS method has been further improved through the method SWATH LC-MS. SWATH is a data independent acquisition form of mass spectrometry, where the mass range is partitioned in small mass windows, which are then analysed with tandem MS. The key advantages are the reproducibility for both individual HCP identification and absolute quantification by applying internal protein standards.
