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 than 0.300, sensitive protein staining methods like silver stain evaluate plate washing procedure for proper or colloidal gold. AAE fractionation and separation performance. of the same conditioned media showed ~1000 protein spots reactive in this ELISA. Because the 2. High Dose Hook Effect or poor dilutional linearity may majority of HCPs will show antigenic conservation be observed in samples with very high concentrations of among all strains of CHO this kit should be HCP. High Dose Hook Effect is due to insufficient adequately reactive to the vast majority of HCPs excess of antibody for very high concentrations of HCPs from your transfected cell line. However, there can present in samples upstream in the purification process. be no guarantee that this assay will detect all Samples greater than 1mg/mL may give absorbances proteins or protein fragments from your process. If less than the 100ng/mL standard. It is also possible for you desire a much more sensitive and specific samples to have certain HCPs in concentrations method than western blot to estimate the exceeding the amount of antibody for that particular coverage of the antibodies in this kit to your HCP. In such cases the absorbance of the sample at all individual HCPs Cygnus Technologies is pleased dilutions may be lower than the highest standard in the to offer AAE as a service. kit, however these samples will fail to show acceptable • Certain sample matrices may interfere in this dilutional linearity/parallelism as evidenced by an assay. The standards used in this kit attempt to apparent increase in dilution corrected HCP simulate typical sample protein and matrices. concentration with increasing dilution. High Dose Hook However the potential exists that the product itself and poor dilutional linearity are most likely to be or other components in the sample matrix may encountered from samples early in the purification result in either positive or negative interference in process. If a hook effect is possible, samples should also this assay. High or low pH, detergents, urea, high be assayed diluted. If the HCP concentration of the salt concentrations, and organic solvents are undiluted sample is less than the diluted sample this some of the known interference factors. It is may be indicative of the hook effect. Such samples advised to test all sample matrices for interference should be diluted at least to the minimum required by diluting the 100ng/mL standard, 1 part to 4 dilutions (MRDs) as established by your qualification parts of the matrix containing no or very low HCP studies using your actual final and in-process drug impurities. This diluted standard when assayed as samples. The MRD is the first dilution at which all an unknown, should give an added HCP value in subsequent dilutions yield the same HCP value within the range of 15 to 25 ng/mL. Consult Cygnus the statistical limits of assay precision. The HCP value to Technologies Technical Service Department for be reported for such samples is the dilution corrected advice on how to quantitate the assay in value at or greater than the established MRD. The problematic matrices. diluent used should be compatible with accurate • Avoid the assay of samples containing sodium recovery. The preferred diluent is our Cat# I028 azide (NaN3) which will destroy the HRP activity of available in 100mL, 500mL, or 1 liter bottles. This is the the conjugate and could result in the under- same material used to prepare the kit standards. As the estimation of HCP levels. sample is diluted in I028, its matrix begins to approach that of the standards, thus reducing any inaccuracies Assay Protocol caused by dilutional artifacts. Other prospective diluents must be tested for non-specific binding and recovery by • The HRP conjugate will have a cloudy using them to dilute the 100ng/mL standard, as appearance. This is normal and does not indicate described in the “Limitations” section below. contamination. Overtime, you may observe a
slight precipitate. This precipitate is Limitations inconsequential to assay results. We suggest a simple inversion of the bottle to re-suspend it. • Before relying exclusively on this assay to detect • The assay is very robust such that assay variables host cell proteins, each laboratory should qualify like incubation times, sample size, and other that the kit antibodies and assay procedure yield sequential incubation schemes can be altered to acceptable specificity, accuracy, and precision. A manipulate assay performance for more suggested protocol for this qualification can be sensitivity, increased upper analytical range, or obtained from our Technical Services Department reduced sample matrix interference. Before or our web site. modifying the protocol from what is • The standards used in this assay are comprised of recommended, you are advised to contact CHO HCPs obtained after the culture of null CHO Technical Services for input on the best way to cells in protein free culture media. 1D Western blot achieve your desired goals. analysis of the antibodies used in this kit • The protocol specifies use of an approved orbital demonstrates that they recognize the majority of microtiter plate shaker for the immunological distinct PAGE separated bands seen using steps. These can be purchased from most
800-F550, Rev. 4, 27JUN2018 CHO HCP 3G ELISA Product Insert 3
laboratory supply companies. If you do not have Assay Protocol such a device, it is possible to incubate the plate without shaking however it will be necessary to 1. Pipette 100L of anti-CHO:HRP (#F551) into each extend the immunological incubation step in the well.. plate by about one hour in order to achieve comparable results to the shaking protocol. Do 2. Pipette 50L of standards, controls and samples into not shake during the 30 minute substrate wells indicated on work list. incubation step, as this may result in higher backgrounds and worse precision. 3. Cover & incubate on orbital shaker at 400 - 600rpm • Bring all reagents to room temperature. Set-up for 2 hours at room temperature, 24C + 4C. plate spectrophotometer to read dual wavelength at 450nm for the test wavelength and ~650nm for 4. Dump contents of wells into waste. Blot and gently but firmly tap over absorbent paper to remove most of the reference. the residual liquid. Overly aggressive banging of the • Thorough washing is essential to proper plate or use of vacuum aspiration devices in an attempt performance of this assay. Automated plate to remove all residual liquid is not necessary and may washing systems or other vacuum aspiration cause variable dissociation of antibody bound material devices are not recommended. The manual resulting in lower ODs and worse precision. Fill wells method described in the assay protocol is generously to overflowing with diluted wash solution preferred for best precision, sensitivity and using a squirt bottle or by pipetting in ~350µL. Dump accuracy. A more detailed discussion of this and tap again. Repeat for a total of 4 washes. Wipe off procedure can be obtained from our Technical any liquid from the bottom outside of the microtiter Services Department or on our web site. In wells as any residue can interfere in the reading step. addition, a video demonstration of proper plate Do not allow wash solution to remain in wells for longer washing technique is available in the ‘Technical than a few seconds. Do not allow wells to dry before Help’ section of our web site. adding substrate.
• All standards, controls, and samples should be 5. Pipette 100L of TMB substrate (#F005). assayed at least in duplicate.
• Maintain a repetitive timing sequence from well to 6. Incubate at room temperature for 30 minutes. DO well for all assay steps to ensure that all NOT SHAKE. incubation times are the same for each well. • Make a work list for each assay to identify the 7. Pipette 100L of Stop Solution (#F006). location of each standard, control, and sample. • It is recommended that your laboratory assay 8. Read absorbance at 450/650nm. appropriate quality control samples in each run to ensure that all reagents and procedures are correct. You are strongly urged to make Example Data controls in your typical sample matrix using Abs. HCPs derived from your cell line. These Mean Well # Contents at 450- controls can be aliquoted into single use vials Abs. and stored frozen for long-term stability. 650nm A1 Zero Std 0.122 • If the substrate has a distinct blue color prior to 0.125 assay it may have been contaminated. If the B1 Zero Std 0.128 absorbance of 100L of substrate plus 100L of C1 1ng/mL 0.167 0.169 stop against a water blank is greater than 0.1 it D1 1ng/mL 0.171 may be necessary to obtain new substrate or the E1 3ng/mL 0.253 0.250 sensitivity of the assay may be compromised. F1 3ng/mL 0.246 • Strips should be read within 30 minutes after G1 12ng/mL 0.571 0.575 adding stop solution since color will fade over H1 12ng/mL 0.579 time. A2 40ng/mL 1.450 1.436 B2 40ng/mL 1.422 C2 100ng/mL 2.709 2.722 D2 100ng/mL 2.734
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Calculation of Results Sensitivity
The standards may be used to construct a standard The lower limit of detection (LOD) is defined as that curve with values reported in ng/mL “total immuno- concentration corresponding to a signal two standard reactive HCP equivalents”. This data reduction may be deviations above the mean of the zero standard. LOD is performed through computer methods using curve-fitting ~0.3 ng/mL. routines such as point-to-point, cubic spline, or 4 The lower limit of quantitation (LOQ) is defined as the parameter logistic fit. Do not use linear regression lowest concentration, where concentration coefficients of analysis to interpolate values for samples as this variation (CVs) are less than 20%. The LOQ is ~1 may lead to significant inaccuracies! Data may also ng/mL. be manually reduced by plotting the absorbance values of the standard on the y-axis versus concentration on Specificity/Cross-Reactivity the x-axis and drawing a smooth point-to-point line. Absorbances of samples are then interpolated from this Western blot and ELISA analysis against 2 commercial standard curve. CHO cell lines identified as CHO-S and K1 indicate that most of the proteins are conserved among all lines. Therefore this assay should be useful for detecting Quality Control HCPs from other CHO strains. Western blot, both 1 & 2 dimensional, is highly orthogonal to ELISA and to non- • Precision on duplicate samples should yield specific protein staining methods such as silver stain or average % coefficients of variation of less than colloidal gold. As such, the lack of identity between 10% for samples in the range of 3-100ng/mL. CVs silver stain and western blot does not necessarily mean for samples less than 3 ng/mL may be greater there is no antibody to that protein or that the ELISA will than 10%. not detect that protein. If you desire a much more • It is recommended that each laboratory assay sensitive and specific method than Western blot to appropriate quality control samples in each run to estimate the coverage of the antibodies in this kit to your ensure that all reagents and procedures are individual HCPs Cygnus Technologies is pleased to correct. offer AAE as a service. This method has been shown to be at least 100 fold more sensitive than Western blots in Performance Characteristics detecting antibody reactivity to individual HCPs. The same antibody as is used for both capture and HRP label can be purchased separately. Cygnus Technologies has qualified this assay by Cross reactivity to non-HCP components has not been conventional criteria as indicated below. A copy of this extensively investigated with this kit. You should qualification report can be obtained on our web site or evaluate components in your samples for positive by request. This qualification is generic in nature and is interferences such as cross reactivity and non-specific intended to supplement but not replace certain user and binding. Negative interference studies are described product specific qualification and qualification that below. should be performed by each laboratory. At a minimum each laboratory is urged to perform a spike and recovery study in their sample types. In addition, any of your Precision samples types containing process derived HCPs within or above the analytical range of this assay should be Both intra (n=20 replicates) and inter-assay (n=10 evaluated for dilutional linearity to ensure that the assay assays) precision were determined on 3 pools with low is accurate and has sufficient antibody excess for your (~3ng/mL), medium (~12ng/mL), and high particular HCPs. Each laboratory and technician should concentrations (~60ng/mL). The % CV is the standard also demonstrate competency in the assay by deviation divided by the mean and multiplied by 100. performing a precision study similar to that described Intra assay Inter assay below. A more detailed discussion of recommended Pool user qualification protocols can be obtained by CV CV contacting our Technical Services Department or at our Low 6.9% 7.2% web site. Medium 2.7% 4.4% High 3.7% 5.0%
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Recovery/Interference Studies Ordering Information/ Customer Service Various buffer matrices commonly used in purification of therapeutic proteins and monoclonal antibodies as well Cygnus Technologies also offers kits for the extraction as in-process and final formulation drug substances and detection of CHO Host Cell DNA. The following kits were evaluated by adding known amounts of CHO HCP are available: preparation used to make the standards in this kit. All of these samples yielded acceptable recovery defined as • Residual Host Cell DNA extraction: between 80-120%. The standards used in this kit contain 8mg/mL of bovine serum albumin intended to Cat # D100W, DNA Extraction Kit in 96 deep well plate simulate non-specific protein effects of most sample Cat # D100T, DNA Extraction Kit in microfuge tubes proteins. However, very high concentrations of some products may interfere in the accurate measurement of • Extraction and PCR amplification of CHO Host Cell HCPs. In general, extremes in pH (less than 5.0 and DNA for use with user supplied master mix: greater than 8.5), high salt concentration, high Cat # D555W, DNA Extraction Kit in 96 deep well plate polysaccharide concentrations, urea, organic solvents, Cat # D555T, DNA Extraction Kit in microfuge tubes and most detergents can cause under-recovery. Each user should qualify that their sample matrices yield • Residual CHO Host Cell DNA extraction and accurate recovery. Such an experiment can be detection using PicoGreen dye: performed, by diluting the 100ng/mL standard provided with this kit, into the sample matrix in question as Cat # D550W, DNA Extraction Kit in 96 deep well plate described in the “Limitations” section. Cygnus Cat # D550T, DNA Extraction Kit in microfuge tubes Technologies offers a more concentrated form of the HCP used to prepare the kits standards for your spike To place an order or to obtain additional product recovery and preparation of analyte controls. information contact Cygnus Technologies:
Hook Capacity www.cygnustechnologies.com Cygnus Technologies, LLC Increasing concentrations of HCPs greater than 100 4332 Southport Supply Rd. SE ng/mL were assayed as unknowns. The hook capacity, Southport, NC 28461 USA defined as that concentration yielding an absorbance Tel: 910-454-9442 reading less than the 100 ng/mL standard was ~1 Fax: 910-454-9443 mg/mL. Email: [email protected] ______
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