CHO Host Cell Proteins 3rd Generation Immunoenzymetric Assay for the Measurement of CHO Host Cell Proteins Catalog # F550 Intended Use immunoenzymetric assay (ELISA) method employed in this kit overcomes the limitations of Western blots This kit is intended for use in determining the presence providing on the order of 100 fold better sensitivity. This of host cell protein impurities in products manufactured simple to use, objective, and semi-quantitative ELISA is by expression in the CHO cell line. The kit is for a powerful method to aid in optimal purification process Research and Manufacturing Use Only and is not development, process control, routine quality control, intended for diagnostic use in humans or animals. This and product release testing. This kit is “generic” in the is the 3rd Generation ELISA kit for CHO HCP detection. sense that it is intended to react with essentially all of the Cygnus Technologies continues to market two earlier HCPs that could contaminate the product independent generation kits, Cat #s F015 and CM015 for CHO HCP of the purification process. The antibodies have been detection. While those kits are broadly reactive to CHO generated in goats and affinity purified using CHO HCPs HCPs among all strains and have been successfully found in protein free conditioned media. Western blot, qualified for a range of drug substances, this 3rd both 1 & 2 dimensional, was used as a preliminary Generation assay uses an even more broadly reactive method and established that the antibodies reacted to antibody. The 3rd Generation Cat# F550 kit offers the majority of HCP bands resolved by the PAGE greater sensitivity and a more linear standard curve separation. Further characterization of coverage of the while incorporating a number of improvements to antibodies to the CHO CHP standards was overcome matrix interference and sample dilutional accomplished by a immunoaffinity chromatography linearity problems sometimes encountered in the two method we term Antibody Affinity Extraction (AAE). This older kits. method is superior to 2D Western blot and terms of sensitivity, specificity, and predictability of how the antibodies perform in the ELISA. The AAE fractionation Summary and Explanation of conditioned media derived HCP from 2 different CHO strains (CHO-S and K1) showed reactivity to ~1000 Expression of therapeutic proteins in CHO cells is a cost HCPs. For more information on AAE please contact our effective method for production of commercial quantities Technical Services department. of a drug substance. The manufacturing and purification Special procedures were utilized in the generation of process of these products leaves the potential for these antibodies to ensure that low molecular weight impurities by host cell proteins (HCPs) from CHO cells. and less immunogenic impurities as well as high Such impurities can reduce the efficacy of the molecular weight components would be represented. As therapeutic agent and result in adverse toxic or such, this kit can be used as a process development tool immunological reactions and thus it is desirable to to monitor the optimal removal of host cell impurities as reduce HCP impurities to the lowest levels practical. well as in routine final product release. Immunological methods using antibodies to HCPs This highly sensitive ELISA kit has been qualified for such as Western Blot and ELISA are conventionally testing of final product HCPs using actual in-process and accepted. While Western blot is a useful method aiding final drug substance samples from 5 different drug in the identity of HCPs, it suffers from a number of products including monoclonal antibodies and other limitations. Western blot is a complex and technique therapeutic proteins. The assay had sensitivity to detect dependent procedure requiring subjective interpretation HCP in all 5 final drug substances showing acceptable of results. Furthermore, it is essentially a qualitative dilutional linearity and spike recovery. Based on this method and does not lend itself to obtaining quantitative experience this assay should have application as a answers. The sensitivity of Western blot is severely multi-use assay for other products expressed in CHO limited by the volume of sample that can be tested, and cells. Each user of this kit is encouraged to perform a by interference from the presence of high concentrations similar qualification study to demonstrate it meets their of the intended product. While Western Blot may be able analytical needs. Provided this kit can be satisfactorily to detect HCPs in samples from upstream in the qualified for your samples, the application of a more purification process, it often lacks adequate sensitivity process specific assay may not be necessary in that and specificity to detect HCPs in purified downstream such an assay would only provide information redundant and final product. The microtiter plate to this generic assay. However, if your qualification Materials & Equipment Required studies indicate the antibodies in this kit are not sufficiently reactive with your process specific HCPs it But Not Provided may be desirable to also develop a more process specific ELISA. This later generation assay may require • Microtiter plate reader spectrophotometer with the use of a more specific and defined antisera. dual wavelength capability at 450 & 650nm. (If Alternatively, if the polyclonal antibody used in this kit your plate reader does not provide dual provides sufficient sensitivity and broad antigen wavelength analysis you may read at just the reactivity, it may be possible to substitute the standards 450nm wavelength.) used in this kit for ones made from the impurities that • Pipettors - 50L and 100L typically co-purify through your purification process and • Repeating or multichannel pipettor - 100L thus achieve better accuracy for process specific HCPs. • Microtiter plate rotator (400-600 rpm) The use of a process specific assay with more defined • Sample Diluent (recommended Cat # I028) antigens and antibodies in theory may yield better • Distilled water specificity, however such an assay runs the risk of being • 1 liter wash bottle for diluted wash solution too specific in that it may fail to detect new or atypical impurities that might result from some process Storage & Stability irregularity or change. For this reason it is recommended that a broadly reactive “generic” host cell protein assay be used as part of the final product purity analysis even • All reagents should be stored at 2C to 8C for when a process specific assay is available. If you deem stability until the expiration date printed on the kit. a more process specific assay is necessary, Cygnus • Reconstituted wash solution is stable until the Technologies is available to apply its proven expiration date of the kit. technologies to develop such antibodies and assays on • After prolonged storage, you may notice a salt custom basis. precipitate and/or yellowing of the wash concentrate. These changes will not impact assay Principle of the Procedure performance. To dissolve the precipitate, mix the wash concentrate thoroughly and dilute as The CHO assay is a two-site immunoenzymetric assay. directed in the ‘Preparation of Reagents’ section. Samples containing CHO HCPs are reacted Reconstituted wash solution is stable until the simultaneously with a horseradish peroxidase (HRP) expiration date of the kit. enzyme labeled anti-CHO antibody (goat polyclonal) in microtiter strips coated with an affinity purified capture anti-CHO antibody. The immunological reactions result Precautions in the formation of a sandwich complex of solid phase antibody-HCP-enzyme labeled antibody. The microtiter • For Research or Manufacturing use only. strips are washed to remove any unbound reactants. • Stop reagent is 0.5M H2SO4. Avoid contact with The substrate, tetramethylbenzidine (TMB) is then eyes, skin, and clothing. reacted. The amount of hydrolyzed substrate is read on • This kit should only be used by qualified a microtiter plate reader and is directly proportional to technicians. the concentration of CHO HCPs present. Preparation of Reagents Reagents & Materials Provided Component Product # • Bring all reagents to room temperature. Anti-CHO:HRP F551 • Dilute wash concentrate to 1 liter in distilled water, Affinity purified goat antibody conjugated to HRP label with kit lot and expiration date, and store at in a protein matrix with preservative. 1x12mL 4C. Anti-CHO coated microtiter strips F552* 12x8 well strips in a bag with desiccant Procedural Notes CHO HCP Standards F553 CHO HCPs in bovine serum albumin with preservative. Standards at 0, 1, 3, 12, 40, and 1. Complete washing of the plates to remove excess 100ng/mL. 1 mL/vial unreacted reagents is essential to good assay Stop Solution F006 reproducibility and sensitivity. We advise against the use 0.5M sulfuric acid. 1x12mL of automated or other manually operated vacuum TMB Substrate F005 aspiration devices for washing plates as these may 3,3’,5,5’ Tetramethylbenzidine. 1x12mL result in lower specific absorbances, higher non-specific Wash Concentrate (20X) F004 absorbance, and more variable precision. The manual Tris buffered saline with preservative. 1x50mL wash procedure described below generally provides *All components can be purchased separately except # F552. lower backgrounds, higher specific absorbance, and better precision. If duplicate CVs are poor, or if the 800-F550, Rev. 4, 27JUN2018 CHO HCP 3G ELISA Product Insert 2 absorbance of the ‘0’ standard is greater