Goat Anti-HEK 293 HCP Polyclonal Antibody

Host cell proteins (HCPs) are impurities associated with the protein production process. They are expressed by the host cells used to produce biopharmaceutical proteins and are a mixture of several different proteins. During purification of the target protein, most HCPs are removed (>99%), but some HCPs are still co-purified with the product and remain in the product. These residual HCPs are potentially harmful and may interfere with drug action or cause allergic reactions. Therefore, it is necessary to characterise and quantify HCPs in APIs and Downstream Purification Process (DSP) intermediates. HCP residues are an important assessment indicator for process stability monitoring and a key quality control indicator for recombinant vaccines and recombinant antibody drugs. The level of HCP residues in biological products is generally considered to be a critical quality attribute (CQA) of the product. National regulatory authorities such as the FDA and EMA require that biopharmaceuticals be analysed and purified to reduce host cell protein HCPs to acceptable levels. Acceptability of HCP is assessed on a case-by-case basis and depends on several factors including: dose, frequency of administration, type of drug and severity of disease. US FDA recommended level: 1-100ppm (ppm: one part per million).

Figure 1. Workflow used to characterize the quality attributes of mAbs and identify HCPs in the culture supernatants. (Sources: Park JH, et al. 2017)

With the development of biotechnology and the globalization of bioinformation, a large number of biological products have been produced by cells. Therefore, establishing appropriate HCP detection methods will help control the quality of biological products and improve the safety of biological products. In 2015, the United States Pharmacopeia (USP) issued a general chapter: <1132> Residual Host Cell Protein Measurement in Biopharmaceuticals. The general chapter describes the important role of electrophoresis, enzyme-linked immunosorbent assay (ELISA) and LC-MS/MS in HCP detection. Even small amounts of host cell proteins (HCPs) in the final drug can produce an immunogenic response; therefore, testing of products and process intermediates is required. Traditional protein detection methods, such as high-performance liquid chromatography and total protein staining, do not have the sensitivity and specificity required for HCP detection. Optimized immunoassay (ELISA) and mass spectrometry (MS) methods are more suitable for the measurement and characterization of HCPs. There are now a variety of analytical techniques to understand the presence and abundance of HCPs in bioprocesses, as follows: Enzyme-linked immunosorbent assay (ELISA); this method is a routine method used in many pharmacopoeias to measure total HCP concentrations, and it has the characteristics of high efficiency and convenience. However, in this assay, there may be a phenomenon of missing detection of non-immunoreactive or weakly immunoreactive proteins in animals; in addition, this method cannot be used to define which HCP components are present, which shows the limitations of this method. 2D-PAGE/2D-Western blot; This is a method that separates HCPs into single components through gel, which can mainly perform qualitative analysis on the composition of HCPs, but the limitation of this method is that there will be a situation where high-abundance protein spots block low-abundance protein spots (i.e., they are not completely separated), resulting in inaccurate analysis. In addition, the sensitivity depends on different staining techniques. 2D-PAGE combined with protein blotting can also identify specific HCPs, but this method has low throughput and is time-consuming. Liquid chromatography tandem mass spectrometry (LC-MS/MS); Mass spectrometry can detect the presence of many proteins in the same sample. This method can provide a certain degree of quantification and compare the protein content in different samples. However, in highly purified recombinant protein samples, residual HCP detection requires highly sensitive instruments and skilled operators, and the presence of a large amount of recombinant protein can be complicated compared to low-level HCPs. This method still cannot fully detect all HCPs present. General Chapter <1132> focuses on the application of immunoassay technology represented by ELISA in HCP detection. ELISA is characterized by simple operation, rapidity, and high throughput, and is considered the gold standard for HCP residue detection. Since there are thousands of HCP antigens and corresponding antibodies, and the types and numbers of HCPs decrease with the purification steps, this poses a challenge to the validation of HCP immunoassay methods. For the validation of intermediate process samples, the focus is on method accuracy (spiked recovery), dilution linearity and precision. The final product should be examined from multiple aspects such as accuracy, precision, dilution linearity, specificity, and quantitation limit. The common HCP ELISA analytical method development plan should be determined according to the different stages of the project.
Although none of the above single methods can accurately and efficiently determine HCP, different detection technologies can be combined. For example, combining ELISA with 2D-PAGE and using immunoblotting technology can verify the coverage of antibodies in ELISA, which helps to improve the accuracy of the ELISA method. 2D-PAGE can also be combined with LC-MS/MS to identify specific HCP components, which helps to identify high-risk factors. In the early stages of development, general commercial kits are acceptable for detecting HCPs, but once biopharmaceuticals enter the Phase III clinical trial stage, due to limitations such as insufficient antibody coverage of general kits, a validated, product-specific HCP detection method is required. Developing such a product-specific detection method is a time-consuming process involving multiple steps, including antiserum production, antibody purification, establishment and optimization of ELISA methods, confirmation and validation.

Host Cell-Derived Proteins
Process-Related Impurities
Host Protein Impurities
Residual Host Proteins
Endogenous Host Proteins
Host-Derived Contaminants
HCP Contaminants