Recombinant expression by the C1 filamentous fungus Thermothelomyces heterothallica cell line is a simple and cost-effective method for production of monoclonal antibodies, recombinant proteins, and vaccines. The manufacturing and purification process of these products leaves the potential for impurities by host cell proteins (HCPs) from C1 cells. HCP can reduce the efficacy of the therapeutic agent, result in adverse immunological reactions or interfere with drug stability. Thus, HCP impurities should be reduced to the lowest levels practical. This simple-to-use, objective, and semi-quantitative ELISA is a sensitive and specific method to aid in optimal purification process development, process control, and in routine quality control and product release testing. This kit is “generic” in the sense that it is intended to react with essentially all of the HCPs that could contaminate the product independent of the purification process. The antibodies have been generated against and affinity purified using C1 HCPs recovered from protein additive free C1 growth media. The antibodies used in this kit were characterized by Antibody Affinity Extraction (AAE) and Mass Spectrometry, demonstrating reactivity to the majority of HCPs. This new Cygnus Technologies' C1 HCP ELISA Kit provides the specificity and sensitivity (LOD ~1 ng/ml, LLOQ ~3 ng/ml) to detect HCPs with reproducibility that supports regulatory compliance, aiding in optimal purification process development, process control, routine quality control, and product release testing. Please use Sample Diluent, Item No. I028, with these kits; available separately
The assay protocol recommended in the kits' product insert have been developed with consideration given to typical limits of detection required in bioprocess impurity analysis. The assays are very robust with respect to protocol options and thus it is possible to modify the protocol to achieve performance parameters more optimal for your analytical needs. Sample volume, incubation times, and use of various sequential (forward or reverse sequential) schemes can lead to significant changes in sensitivity, reduction of non-specific sample matrix affects, or greater upper analytical range. If you change the protocol, it will be necessary to qualify that those changes achieve acceptable accuracy, specificity, and precision. Please refer to the section below on "How to Qualify HCP & Impurity Assays." For advice on how best to modify an assay to meet your objectives you are encouraged to contact our Technical Services Department.
We manufacture our kits for lot-to-lot consistency and restrict and evaluate changes in any components or procedures that could impact accuracy in the customers' laboratory. However, for generic kits, Cygnus performs QC on a limited range of parameters that may or may not be sensitive to your product specific issues. The routine run-to-run quality control of ELISA is best accomplished by assaying control samples across the important analytical range of the assay. We recommend 2 to 3 controls, a low control in the range of about 2 to 4 times the assay Limit of Quantitation (LOQ), a medium control, and a high control. Ideally, these controls should be made using your source of analyte (e.g. HCPs from your process). Furthermore, the controls should be in the same matrix as your critical samples or after dilution to MRD for your final drug substance. By using your source of analyte in your sample matrices, you will be best able to identify any problems within the run or between runs and kit lots. Cygnus offers Control Sets for several ELISA kits including; CHO, HEK, E.coli, PG13, and Protein A. For more information on Control Sets or advice on how to make and establish controls specific to your needs please contact technical support.
Use of laboratory-specific controls is the only way to assure total quality control of the assay for your needs. Controls should be made in bulk, aliquoted for single use, and frozen at -80°C until stability studies indicate some other storage conditions are adequate. Once you have statistically established a range for these samples, they will become your most sensitive and specific tool to assure quality control of the assay. Do not rely on curve fit parameters as quality control specifications in the absence of true analyte controls. Curve fit parameters such as R square, slope, y-intercept, and upper and lower asymptotes are not sensitive or specific enough to reliably detect assay problems. Use of such parameters in the absence of true analyte controls, will frequently yield erroneous results. Contact our Technical Services Department for advice on how to make and establish controls specific to your needs.
Numbers of Replicates - When precision is very good (average replicate %CV on ODs are less than 5%) we feel duplicate analysis is adequate and the most effective approach. In the case of duplicate analysis, we do not allow for editing of any apparent outliers since there is no statistical basis for establishing which of the duplicates is inappropriate. Thus, in duplicate analysis we suggest repeating any sample that yields a %CV greater than 20%.
Many of our customers have expressed an interest in automating our ELISAs to improve throughput and assay performance. Cygnus Technologies has much experience with various automation platforms. These systems include automated microtiter plate instruments and other liquid handling systems as well as several “biosensor” platforms. Using our proven and well-qualified antibodies and reagents in a biosensor platform still presents your lab with significant challenges in re-qualifying and optimizing the reagents. Before considering adapting our reagents to an automated system, you are encouraged to contact our Technical Service Department for guidance on how to approach assay development, qualification, and validation.
The promises of automation are higher throughput, faster turnaround of results, and improved performance in terms of parameters like sensitivity and precision. The practical ability of the many systems now being marketed to meet these promises is variable. Before embarking on a quest for automation, we urge you to contact our Technical Services Department. After evaluating your testing needs we can guide you to the system that best meets your requirements.
Cygnus kits and reagents have been formulated to withstand shipment and/or temporary storage under ambient temperature conditions without any significant deterioration. Orders within North America are shipped next day delivery service without cold packs. International orders are shipped with cold packs to protect from extreme heat. Although stability studies have demonstrated that Cygnus kits can be held several weeks at ambient temperatures without any significant deterioration, we recommend storage at 2-8°C upon receipt of the products. Our components have been challenged at elevated temperature excursions, addressing concerns over extreme summertime temperatures, delays in shipping, custom clearances and/or refrigeration problems at the customer locations.
Should you have concerns regarding the performance of kits that have been subject to temperature deviations, we recommend an analysis of your control samples and historical specifications that determine the integrity of the kit. If the curve absorbances, sensitivity, and controls are within range, then the kit is acceptable for use.
Some samples, particularly those from upstream in your purification process will have impurity analyte concentrations above the analytical range of our very sensitive ELISA kits. Such samples may require very large dilutions in order to overcome “Hook Effect” and to achieve acceptable “sample dilution linearity”. In addition to the “Hook Effect,” the matrix of some samples may interfere non-specifically with the assay and also result in under recovery of the true analyte levels. Simple dilution of those samples is often adequate to buffer out such interference provided the dilution does not reduce analyte concentrations below the Limit of Quantitation (LOQ) of the assay. In cases where dilution of your samples is not an option contact our Technical Service Departmentfor advice on how best to overcome sample matrix interference.
Cygnus offers assay specific diluents for each of its kits. The catalog numbers for these diluents can be found in the kit product insert or by contacting Customer Service. We strongly recommend use of those diluents because they are the same formulation as the matrix used for the kit standards. Thus, as you dilute your samples in our diluent, your sample matrix begins to approach that of the standards and in this way greatly minimizes any dilution artifacts that could occur if you were to use another diluent.
If you elect to use another diluent you must validate that it provides accurate results. We recommend two critical experiments for validation:
First, you must assay the diluent alone to determine that it does not yield absorbance values significantly above or below the absorbance for the kit zero standard. OD values above the zero standard indicate that the diluent may have low levels of the analyte or that the diluent causes an increase non-specific binding, relative to the kit standards matrix. OD values less than the kit standard may indicate that the proposed diluent is lowering non-specific binding and may also be inhibiting specific binding. In any event use of a diluent that does not match the standards risks significant errors when the sample concentration gets multiplied by the dilution factor.
Second, you must perform a spike & recovery experiment into your proposed diluent at several levels across the analytical range of the assay. For a diluent to be deemed acceptable we suggest a recovery specification of 95% to 105%. In general, the formulation of diluents should be a neutral pH with some carrier protein added to block non-specific adsorptive losses of the analyte. Use of just PBS or TBS without a carrier protein can be problematic because the analyte diluted in the range of the assay (ng/mL) can very significantly adsorb to the dilution tube resulting in low recovery!
Your diluents should not have sodium azide as a preservative nor significant detergent concentrations as these will reduce assay accuracy.
