Assay Qualification Template for Host Cell Protein ELISA

Transcription

1 Assay Qualification Template for Host Cell Protein ELISA Introduction: With ever increasing importance being placed on host cell protein (HCP) removal during the purification process, it is critical to ensure that the selected HCP ELISA method is fit for its intended use. From a regulatory perspective, a well developed and qualified HCP ELISA will ensure that HCPs have been reduced to safe levels and that the purification process is consistent from batch to batch. From the process development and manufacturing perspective a good HCP ELISA will help guide purification process decisions to ensure the maximum level of HCP clearance. The HCP ELISA can also play a key role in identifying purification process failures. Summary: This assay qualification template describes the minimum experimentation that should be performed to demonstrate that a HCP ELISA is fit for use. This includes the large format - dimensional gel and Western blot analysis, dilutional linearity analysis, and the determination of accuracy and precision. Additional qualification experiments can be performed to more fully define the assay such as LLOQ and LOD determination, reproducibility, robustness, specificity, linearity, and range. Disclaimer: This HCP Assay Qualification Template reflects the regulatory environment and requirements at the time of this writing, February 0. The Template will help to demonstrate that an assay is acceptable to use in the early stages of a project. This Template is not a substitute for a more comprehensive assay validation which must be performed prior to a Phase III filing. It is always a good idea to keep an open discussion with regulators to determine what is expected before filing an IND. Preliminary Evaluation of ELISA: It is advisable to perform a preliminary evaluation of any HCP ELISA prior to conducting a comprehensive assay qualification. The ELISA should be able to demonstrate clearance of HCP through the purification process with good sensitivity. Simple testing can be performed upfront to quickly assess if the candidate HCP ELISA can meet this goal. If the ELISA can detect HCP in a valid manner then the assay is a candidate for qualification. If the ELISA fails to detect HCP downstream or in the final Drug Substance, a more sensitive ELISA should be considered. Preliminary Assessment of ELISA: Prepare an initial dilution of the Drug Substance to a protein concentration of <5 mg/ml in a suitable assay diluent. Make three additional -fold dilutions in the assay diluent, and test in the ELISA along with all assay standards and controls. At this stage it is not necessary to evaluate spike recovery. Evaluation of the Data: The goal of this preliminary test is to determine if the HCP ELISA can detect HCP above the Limit of Detection (LOD) and preferably above the Lower Limit of Quantification (LLOQ) in final Drug Substance or immediately preceding the process purification step. Therefore, it is best to set up the analysis as a threshold/limits test.

2 If the test result is higher than the LLOQ, the assay is a good candidate for assay qualification. If the test result is greater than the LOD but lower than the LLOQ, the assay is a candidate for qualification. While the assay may or may not be suitable for a final release test, it will be useful for purification process optimization. If the result is less than the LOD of the assay we recommend evaluating additional HCP ELISAs. Test upstream samples to determine if the assay has utility as a process development tool. Failure to detect HCP in final Drug Substance could mean either a very clean product or that a more product process specific HCP antibody and assay may be required. Cygnus offers custom antibody and assay development services to meet these requirements. Antibody & ELISA Qualification The essential experiments for qualification are D Western blot, dilutional linearity, spike recovery and precision.. -Dimensional SDS-PAGE/Western Blot Analysis: Overview -Dimensional SDS-PAGE/Western Blot Analysis (D Analysis) is performed to obtain an estimate of the coverage of the ELISA antibody to the total host cell proteins present in the production cell line or null cell line of the same species. There are acknowledged limitations to D Analysis and therefore this testing is performed only to estimate coverage and to achieve a level of assurance that the antibodies chosen are appropriate for use. The final determination that an assay is fit for use should come from the assay qualification data derived from the ELISA. Testing In this analysis, proteins are separated by isoelectric point and molecular weight on duplicate large format gels by SDS-PAGE. One of the gels is silver stained and the other is transferred to a PVDF membrane for Western blotting with the antibody used in the ELISA. The two images should be electronically overlaid and a computer analysis of coverage can be performed. Cygnus Technologies can provide this service if your lab does not have this capability. Presentation of s The most important information that can be derived from the D analysis is the coverage of the ELISA antibody to the total proteins present in the sample. There are important aspects of coverage which must be reviewed to determine suitability of the antibody. First, the coverage of the spots on the Western blot are compared to the spots on the silver stained gel. This number is determined by analyzing which spots on the silver stained gel are present on the western blot. This indicates if the antibody is broadly reactive to the potential HCPs. Second, and equally important, is coverage of the Western blot to all areas of the gel that contain spots on the silver stained gel. In this analysis, the gel should be divided into 4 quadrants. The western blot should have representative coverage in each of the quadrants that contain proteins on the silver stained gel.. al Linearity: Overview All sample types with apparent levels of analyte greater than the LOQ of the assay should initially be evaluated for dilutional linearity as part of assay qualification. This experiment involves performing a series of doubling dilutions within the analytical range of the assay using an approved assay diluent. These

3 dilutions are then assayed and a dilution corrected contaminant concentration is determined at each dilution. This dilutional linearity study establishes freedom from sample matrix interference, and also demonstrates the important condition of antibody excess for the array of contaminants in your samples. If you will be routinely testing in-process samples in addition to final product, the dilutional linearity of each sample type should be determined. This analysis is critical for HCP assays because very high concentrations of certain HCPs may approach saturation of the antibody against that particular HCP. When this happens, there is a risk of significant under-quantitation for that HCP. By performing dilutional analysis one can verify if the antibody is in excess and that the sample matrix itself does not interfere. If the antibody is in a limiting concentration or the sample matrix causes a negative interference, the dilution corrected HCP concentration for a sample increases with increasing dilution. In most cases a dilution will be reached where the dilution corrected value remains essentially constant. We term this the Minimum Required (MRD). Table shows example data where a sample did not yield good dilutional linearity at high concentration, but with additional dilution an MRD was determined at which acceptable dilutional linearity was obtained. In this example, we conclude that the MRD for this in-process sample is :6 and that the concentration of HCP to be reported is 6ng/mL (the average result from dilutions tested above the MRD). Once an MRD is established for a particular sample type, the SOP should reflect that these samples need to be diluted to at least the MRD before assaying. We suggest defining acceptable dilutional linearity as dilution corrected analyte concentrations that vary no more than 80% to 0% between doubling dilutions. Due to the statistical limitations in the low end of the assay range you should avoid consideration of dilutional data where the assay value before dilution correction, falls below the LOQ of the assay. Acceptable diluents may vary from assay to assay and you are encouraged to verify with Cygnus Technologies that your sample diluent is acceptable. Table : Example data of Minimum Required Determination Sample Corrected Value % Change from previous dilution : 46 NA :4 60% :8 4% :6 6 6% : 56 99% : % :8 <LLOQ NA

4 Testing Prepare a -fold dilution of the sample in an appropriate sample diluent (see your kit insert for the recommended sample diluent). Prepare 9 additional -fold dilutions and test in the ELISA. Enter the sample type, dilution factor, and result into Table. Calculations: : of s for the replicates Corrected : x Factor Percentage Difference: (Current Previous ) Previous Table : Example data table for the establishment of dilutional linearity and sample MRD. Sample Replicate Corrected Percentage Difference From Previous NA Presentation of s The dilution at which the percentage difference from the previous dilution is <0% and stays under 0% is considered the MRD for this sample type. All future testing should be performed at this or a higher dilution. If the sample result is below the assay LLOQ before the MRD is achieved, the ELISA may not be suitable for this sample. Contact our Technical Services on how to best proceed if you have dilutional linearity problems.. Accuracy/Spike Recovery: Overview The assessment of accuracy is performed by comparing the recovery of a known amount of HCP spiked into the sample, to the measured recovery in the ELISA. According to current ICH guidelines, accuracy should be assessed using a minimum of 9 determinations over a minimum of concentration levels. This

5 analysis should be performed on all sample types to be tested using the HCP ELISA. All results of the testing should be reported as a percentage recovery. Testing There are two critical decisions that must be made before performing Accuracy testing. The first decision is how much a sample should be diluted prior to testing. If the sample is not diluted enough, it could be below the MRD and the spiked HCP could fall outside of the analytical range of the ELISA. If the sample is diluted too much, the sample matrix essentially becomes sample diluent and the value of the testing is diminished. The second critical decision is the level of spiking solution to be added. If the spiked concentration is too low, the recovery may be difficult to accurately assess. For example, if the sample measures an HCP content of 75ng/mL, a spike concentration of ng/ml is easy to lose in the noise of the ELISA. If a spike concentration is too high, the value of the testing can diminished because the sample result is very small relative to the spike concentration. To keep with the previous example, if the sample result was ng/ml and the spike concentration is 75ng/mL, obtaining a high spike recovery is less dependent on the accuracy of the assay and more on the parameters of the testing. To appropriately ascertain the Accuracy of each sample type, the sample should be diluted to or below the middle of the analytical range of the assay based on the al Linearity experiment. The Spiking Concentration should be set at the midpoint between the middle of the analytical range and the upper asymptote of the standard curve. Prepare independent replicates of the sample to be tested. Each replicate should then be split into a Spiked and an Unspiked sample. Add spike solution to the Spiked tube and mix well. Perform additional -fold dilutions of the Spike and Unspiked sample and test in the ELISA. This will result in 8 individual determinations and a total of 9 results. Table is the plate map that should be used for the Accuracy and Precision testing. The same data set can be used for the Accuracy and Precision calculations. Table : Plate map for Accuracy and Precision testing. A Standard # Unspiked Dil Unspiked Dil Unspiked Dil B Standard # Unspiked Dil Unspiked Dil Unspiked Dil C Standard # Unspiked Dil Unspiked Dil Unspiked Dil D Standard # 4 Spiked Dil Spiked Dil Spiked Dil E Standard # 5 Spiked Dil Spiked Dil Spiked Dil F Standard # 6 Spiked Dil Spiked Dil Spiked Dil G H Positive Control Negative Control Presentation of s Enter the Sample,, Unspiked, & Spike into Table 4. To establish Accuracy, the %Recovery should be 80%-0% of the expected value. The calculations below can be performed to complete the analysis in Table 4.

6 Calculations: Spike Recovery: Spiked result Unspiked Spike Recovery: of Spike Recovery replicates Expected: Known amount of HCP spiked into the sample % Recovery: ( Spike Recovery Expected) 00 Table 4: An example data table for the determination of accuracy of HCP ELISA. Sample Preparation Unspiked Spike Spike Recovery (Spike - Unspiked) Spike Recovery Expected % Recovery Cygnus can furnish an electronic copy of Table 4 to automatically perform the calculations. 4. Precision: Overview: Precision of the method is calculated as the closeness of agreement between multiple measurements of the same homogenous sample. Precision can be measured as Repeatability (closeness of results within a single run), Intermediate Precision (closeness of results from day-to-day or analyst-to-analyst, etc.), and Reproducibility (closeness of results from lab-to-lab). However, for the purposes of a basic assay qualification, this template will only assess assay repeatability. Repeatability expresses precision by making multiple measurements of the source sample within a single run. It is advised to have a second analyst perform the same run on a different day to assess Intermediate Precision. Testing: The same data generated for the Accuracy determination can be used and analyzed in a different way to determine the Precision (repeatability) of the HCP ELISA. This testing will produce 8 individual determinations, results and an overall Intra-assay precision (Repeatability). Presentation of s: Enter the Sample, and Unspiked s into Table 5. The calculations below can be performed to complete the results analysis in Table 5. To establish precision, the Repeatability should be less than 5%.

7 Calculations: : of Unspiked for that dilution Corrected : Unspiked : Corrected Standard Deviation: Standard Deviation of the Corrected %CV: Standard Deviation Unspiked 00 Overall : of all the Corrected s Overall Standard Deviation: Standard Deviation of all the Corrected s Overall %CV (Repeatability): Overall Standard Deviation Overall Unspiked 00 Table 5: An example data table for determination of precision for an HCP ELISA. Sample Preparation Unspiked Corrected Unspiked Standard Deviation % CV Overall Overall Standard Deviation Overall %CV (Repeatability) Conclusions: This template is intended as a guide to establish a standardized method to qualify an HCP ELISA for early stage products/projects. Additional testing can be performed for more assurance, however, the testing outlined in this template should demonstrate that the HCP ELISA selected is fit for use for early project activities, including guiding purification process decisions and ensuring the safety of early clinical material. The Cygnus in-house generic assay qualification data may be used as a substitute for robustness and stability of the reagents, since we view this data as inherent in the method and not something each laboratory needs to perform. However, once the purification process is locked and the project progresses toward Phase III, a full scale assay validation must be performed. For problems, questions, or advice on how to use this template or to validate our assays to detect bioprocess contaminants in your products please call our Technical Services Department.