“Supporting technologies or orthogonal methods are an integral part of how biopharmaceutical manufactures can assure product purity and consistency.”- USP Chapter 1132, 5. SUPPORTING TECHNOLOGIES FOR RESIDUAL HCP DETECTION, IDENTIFICATION, AND MEASUREMENT
Sensitive and specific orthogonal methods are necessary to identify individual host cell protein impurities that persist through your purification process. These orthogonal methods include Antibody Affinity Extraction (AAE™) with 2D-PAGE or MS for HCP antibody coverage analysis and AAE-MS™ for HCP identification and quantitation in in-process samples and final drug substances.
Antibody Affinity Extraction is a method devised by Cygnus Technologies to overcome the technical challenges and limitations of other orthogonal methods such as 2D Western blot and 2D-DIBE in assessing the coverage of a polyclonal antibody to total host cell protein (HCP). In this method the polyclonal antibody is covalently immobilized on a chromatography support. The column is then conditioned to prevent significant leaching of the antibody and to greatly minimize any non-specific binding. The HCP sample in its native, undenatured state is passed over the column for binding and then eluted with acid. The HCP sample is again cycled over the column by binding and elution until no additional HCP is bound. All HCP elution fractions are pooled, buffer exchanged, and concentrated back to the original sample volume. The final sample is then separated by 2D SDS PAGE and analyzed by either a comparison to a silver stain of starting, unextracted sample or by Differential Gel Electrophoresis (DIGE) using Cy3 and Cy5 to label the extracted and starting, unextracted samples.
AAE™ is more predictive of how the anti-HCP Ab will perform in HCP ELISA and provides sufficient sensitivity to evaluate individual HCPs that persist through purification process. Due to the inherent limitations of the 2D WB or 2D-DIBE methods, such as (1) loading capacity, (2) denaturing of native HCP epitopes by harsh sample treatment, (3) failure to transfer some HCPs out of the gel, (4) HCPs bound to the membrane such that antibody binding is sterically inhibited, (5) difficulties in aligning PAGE gel to a WB membrane images, and (6) poor specificity, 2D WB based methods significantly underestimate true Ab coverage to upstream HCPs. More importantly, 2D WB does not predict how that Ab will react to the most important HCPs which are those that co-purify with the drug substance.
AAE-MS approach allows the detection and identification of HCP impurities to help optimize your downstream purification processes and identify any potential HCP impurities in your drug substance (DS). In addition, AAE-MS represents a more objective and direct method to qualify the ELISA as fit for purpose of detecting those HCP that co-purify with your drug substance. AAE-MS can be used for HCP antibody coverage analysis, especially when there is no access to null cell culture, for process monitoring by identifying and quantifying HCPs in process samples, for identification and quantification of hitch-hiker HCPs in the final drug substance, and to gain actionable insights into downstream process development and purification improvements.
Identification and quantification of HCPs by mass spectrometry is a powerful complementary method to ELISA, however drug products often mask HCPs by a factor of 104-106. When combined with AAE as a sample preparation step to enrich HCPs and eliminate most of the DS, MS sensitivity is highly improved.
The integration of data from these orthogonal methods together with ELISA data provide a comprehensive analysis of HCP content, that will meet regulatory expectations. In addition, integration of these advanced orthogonal methods ensures a data-driven approach to understanding whether a generic HCP ELISA is fit for your process monitoring and product lot release or a process-specific assay must be developed.