FDA HCP assays are current the weakest analytical link in the total control strategy for biotech products

Transcription

1 FDA HCP assays are current the weakest analytical link in the total control strategy for biotech products What are the best ways to prepare antigen/antibody reagents with highest HCP coverage? There was no single approach to guarantee complete coverage of HCPs by anti-hcp antibody preparations. Combinations of approaches have been used successfully by sponsors. Various strategies to preparation of the immunogen included using extracellular soluble fractions vs complete cell lysates, using upstream vs downstream material, using upstream and downstream material, using HMW and LMW materials, using material stripped from process chromagraphy steps, using cascade immunization. The pros and cons of different approaches on preparation of the antibodies were discussed that included affinity-purification of all Igs vs affinity purification of only anti-hcp Igs ( active Igs), assessing crossreactivity of the Pabs with the API, assessing the ability to detect low-abundance and/or low MW species (especially in DS). Evaluating and pooling antisera from multiple animals to achieve desired reactivity has also been done. (Not discussed: Pooling antibodies generated in different species for dual-species ELISA )

2 What are the best ways to prepare antigen/antibody reagents with highest HCP coverage? Questions arose regarding the suitability of using HCP material from null construct cultures which may not fully mimic actual process conditions for the expressed proteome, or age of cultures (eg early vs EOP cultures) used for HCP immunogen. Concern was if API has any impact on differential expression and/or co-purification of HCPs, null construct might be deficient or different in HCPs. It was suggested that HCP s from null construct could be compared to HCPs from production to assure comparability of proteins; various comparative methods were mentioned (MS fingerprint, SS gels, 2DIGE). Draft USP general chapter contains substantial technical detail on various ways to produce/characterize HCP immunoreagents based on experiences of committee members. Regulators remained agnostic on technical approaches so long as the reagents are demonstrated to perform as necessary for the assay s intended use (monitor any HCPs that can potentially end up in purified DS) 2D gels/blots are currently the gold standard with regulators although it is acknowledged that they are not perfectly accurate for Ag/Ab specificity (esp if not adequately optimized); encourage supplementation with orthogonal means of assessing immunoreactivuty (AAE, 2D HPLC, etc )

3 How to best replenish these critical reagents? Ideal scenario: Avoid having to replenish such complex mixtures by preparing enough HCP immunogen and anti-hcp Pabs to last through planned product lifetime! If not possible, will need to do sufficient analytical characterization of the components to show comparability. ELISA signal is a sum of the reactions in the wells; the same value can come from different reactions (ie 100 ppm = 2 x 50 ppm each, or 10 x 10 ppm each?). So ELISA alone is not capable of assessing comparabiity of mixed materials (Ag or Ab). Will need to use approaches that can compare the pattern and abundance of HCPs (if that was the change) or the immunospecificity of the Pabs Even process-specific immunoreagents should be assessed for comparable sensitivity and specificity to assure continuity of data with prior reagents (FDA case study: deficient new Pab reagent was not acceptable in assuring continued product quality and consistency for HCPs) (Didn t discuss: How to assure the HCP ref std in the ELISA is suitably reflective of the process HCPs ) Main goal: Producing (by any immunological means necessary) broadly specific HCP immunoreagents, then accurately establishing what they detect vs what they miss of the HCP proteome in production cells. Regulatory acceptance will be driven by the data you provide on your reagents and assays Compendial approaches have different legal implications

4 What is the state-of-the art analytical control strategy for HCP: Are immunoassays still the most suitable method for QC testing of HCPs? Currently HCP ELISAs are the gold standard for QC testing of DS for release (US, HC and EU). MS is very information rich tool on HCPs, but may be too complex of technology for QC; ELISAs generate a summed value for the samples but provides a simple method for operations. How to overcome dilution-related linearity issues in immunoassays with broad HCP coverage? Most challenging aspect of using highly diverse set of Pabs in a single ELISA well. (excellent cartoon). Also seen as hook effect in assay dilution series. Need to experimentally evaluate this for each assay and validate the minimal valid dilution (explained in USP chapter). What other technologies are being used for GMP release testing for HCPs? Other technologies are used for characterization of the QC assay as well as investigation of issues with the product/process (US, HC and EU) Under what circumstances is more than one QC release assay needed to measure HCPs? Specified HCP like PLBL2 that are known to co-purify with DS, others that are present at high abundance in DS (highly abundant milk proteins in DS from transgenic animals swamp out the ELISA total HCP signal), although might be monitored in process vs in QC release (data driven for process control/consistency) What are the characterization assays that support the selection, development, and validation of the QC assay? What is the role of Mass Spectrometry? Clearly MS methodologies are emerging as powerful tools in HCP characterization for process and product, and assessing comparability of process change; investigating problems with HCP assays and HCP immungenicity issues Not clear yet whether it will also become valuable in comparability of originator to biosimilar products

5 Are there common HCPs that should be of particular concern in a given class of products (e.g. Monoclonal Antibodies, Coagulation Factors)? Are there common HCPs in a given cell line that should be specifically checked for when developing a product in that cell line? Maybe so but no common list of these is available (publically) PLBL2 in CHO (soon to be published); others have been published or presented HCP proteiases (several publications) have been shown to have impact on product quality Non-Mab products in human cell line have showed co-purification of HCPs Even from the same cell line different enzymatic protein products showed different hitch-hiker HCPs Biologically, chaperones are high targets for co-purifying proteins Cytokines products should screen DS for co-purifying HCPs that are also cytokines Certain cell culture systems over-express certain host proteins Process affinity resins have been shown to pick up and enrich HCPs Transgenic systems have highly abundant proteins in the HCP mixture that may need special attention to monitoring Novel expression systems (modification of a conventional cell type) have shown specific differences in HCPs (**Can we pull together all of the publically available HCP case study references for citation in the Forum white paper for readers to see?**)

6 Which experiences do the regulators have with HCP characterization, testing, and control that are the basis for their current concerns and expectations? What are the regulators decision processes for evaluating the clinical risk of HCPs? What are the risks with HCPs present in clinical trial batches that initially went undetected? In commercial batches? Finding HCPs in clinical batches after the fact is less risky than finding new HCPs in batches that have never been in clinical trials. What is the regulators thinking about routinely monitoring anti-hcps antibodies during clinical development? CBER: Should develop clinical immunogenicity assay for screening anti-hcp Abs in parallel with the HCP ELISA for product QC testing CDER: ADA immunogenicity assay needed by ph 1 but would require anti-hcp assay on case by case basis (FDA guidance on immunogenciitty risk assessment) EU: 1 year post approval immunogenicity monitoring required (?) What are the regulators current thoughts on acceptable HCP levels: There are NO common HCP limits that can be officially set for all products? Examples were shown where one specific protein caused problems in some products, while lots of HCPs have not been a problem in many products factors include clinical safety, analytical capability, process capability, HCP levels in similar products on the market; Based on target indication for product (dose, route) and purpose (ie liefsaving) CBER: wide range of limits (low ng to low ug) in different units (ppm, ng/mg, ng/u) PEI: Mab target of 1 ng/mg or less has been show to be possible; other therapeutics NMT 100 ppm in early dev; finaized after process val and consistency (eg10 to 30 lots commercial product)

7 How does one determine the suitability of HCP assays at different stages of product lifecycle: During development, when should a change be made from platform/ commercially available assay to the specific HCP assay? How should bridging studies be done between HCP assays? What are the expectations for such studies? When should they be done? What HCP comparability studies should be done to support process changes? At a minimum, assess the specificity of the commercial ELISA as soon as you start using it; may be able to use throughout development esp if supplemented with other chracterizaiton (archive historical samples for retesting with final assay!) Sometimes generic ELISA might not be able to detect the same signals in upstream vs downstream ELISAs ; sometimes difference ELISAs detect different populations in the same products (Sandoz data with originator vs biosimilar) Hindsight has showed using process-specific ELISA as early as possible is best to avoid process dev missing several HCPs; case studies have shown this can have impact on timelines if suddenly finding something not previously detected that is problematic to product that requires process improvements. Can only truly optimize the process steps with accurate measurement of HCPs, otherwise will be making changes with partial data on HCP shifts. Balance risk of missing HCPs by ELISA with power of broad-based reagent independent characterization of process and product (eg MS, 2DIGE)