What are the best ways to prepare antigen/antibody reagents with highest HCP coverage? Regulators remained agnostic on technical approaches so long as the all of the HCP ELISA reagents are demonstrated to perform as necessary for the assay s intended use (monitor any HCPs that can potentially end up in purified DS). Regulatory acceptance of HCP ELISAs will be driven by the data sponsors will provide on your HCP immunoreagents and assays. Draft USP and EP general chapter contain information on various ways to produce/characterize HCP immunoreagents based on experiences of the pharmacopeial HCP committee members. (*SEE SLIDES TMS/KH). It was noted that compendial chapters have different legal implications in different regions of the world, so sponsors should consult their regulatory authorities for any potential impact on their HCP ELISA development and validation strategies. There was no single immunological approach to guarantee complete coverage of total population of HCPs by anti-hcp antibody preparations. Combinations of approaches have been used successfully by numerous sponsors, with major technical improvements made in the last decade. Various strategies debated in the preparation of the HCP immunogen included using complete cellular lysates vs just the extracellular soluble fractions, using upstream or downstream material, using upstream and downstream material, using either or both US/DS material separated into HMW and LMW immunogen fractions, using bound HCPs stripped from process chromagraphy steps, using cascade immunization to enrich anti-hcp Pabs. 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. Elements that might alter the proteome (either the core protein or the PTMs) include age of cultures (eg early vs EOP cultures) as well as the edges of upstream process ranges. Another concern was if API has any impact on differential expression and/or co-purification of HCPs, then a null construct might be deficient in or have a slightly different population of HCPs. To assess the impact (or not) of these elements, it was suggested that HCP populations from different process runs should be analytically compared to de-risk the source(s) of the HCP immunogen. Various comparative methods were mentioned (MS fingerprint, silver or sypro stained gels, 2DIGE comparison of null vs actual cell culture). Several attendees indicated they use multiple types of HCP materials as separate immunogens to allow them to evaluate and pool the most reactive antisera for subsequent puification as the ELISA immunoreagents. It was noted that even if separate materials were used to generate anti-hcp antisera. the HCP ELISA reference standard should be a total HCP mixture (ie reflecting the HCP proteome),.
What are the best ways to prepare antigen/antibody reagents with highest HCP coverage (contt d)? Whether using a single immunogen or separate immunogens, evaluating serum from individual animals than pooling high titer antisera from multiple animals to achieve desired reactivity was reported to have been very successful in producing higher coverage anti-hcp reagents. Mentioned, but not further discussed, was the possibility of generating then pooling antisera generated in different species to produce dual-species HCP immunoreagents. The pros and cons of different approaches on preparation of purified polyclonal antibodies from antisera were also discussed. Currently the two major strategies are 1) affinity-purification of total Igs (ie over Protein A or G affinity columns), or 2) affinity purification of only anti-hcp Igs over HCP columns. The former yields an immunoreagent with only a proportion of specific anti-hcp Pabs and with a higher possibility for cross-reactivity of non-hcp Pabs to the API. The latter yields an active set of anti-hcp Pabs but the Pabs recovered are totally dependent on the nature of the HCP material used on the column (if an HCP wasn t on the column, that anti-hcp Pab won t be in the immunoreagent). Regardless of immunization or purification strategy, both regulators and industry considered it vital to fully characterize the immuno-specificity of the resulting anti-hcp Pab immunoreagent. Key characterization elements were 1) assessing non cross-reactivity with the API protein, 2) assessing the ability to detect low-abundance and/or low HCP MW species, 3) assessing detection of any sub-population of HCPs that are enriched in downstream steps, and 4) demonstrating the percentage of the total HCP proteome of the expression system that the Pab can detect. 2D gels/blots are currently the gold standard with regulators to show the degree of immuno-recognition of the anti-hcp Pabs with the total HCP population. It was acknowledged that 2D gels and blots may miss some HCP Ag/Ab binding reactions because: 1) the gels typically reduce and denature the HCPs, losing any epitopes that are solely conformational (although it was mentioned that conformational-only epitopes could be a small subset of total HCP epitopes), and 2) for each HCP and Pab reagent set, the gel/blot conditions require experimental optimization of each step to achieve accurate results. To supplement 2D gel/blot specificity data, it was encouraged to characterize HCP ELISA reagents with orthogonal means of assessing immunoreactiviy such as 2D HPLC and Antibody Affinity Extraction (AAE). It was noted that since these analytical characterization data sets validate the immuno-specificity of the HCP ELISA, they should be referenced (and are increasingly being included) in HCP ELISA method validation packages. Main goal: Producing (by any immunological means necessary) broadly specific HCP immunoreagents for a given expression system in an given process, then accurately establishing what those immunoreagents detect vs what they miss of the HCP proteome in production cells.
How to best replenish and bridge these critical reagents? It was highly recommended that the preparation and characterization of HCP ELISA reagents (immunogen, antisera and Pabs) are thoroughly documented with sufficient technical details to allow them to be re-created as supplies become depleted.over time in the life cycle of the product. One note made from discussions was that if old in-house HCP ELISA reagents exist from past products, they should be checked for specificity for use in new products. The ideal scenario is to avoid having to replenish such complex protein mixtures by preparing enough HCP immunogen and anti-hcp Pabs to last through planned product lifetime. If this is not possible (eg with ongoing platform processes, or late phase or legacy products), it will be necessary to do sufficient analytical characterization of the immunogens and antibodies to show comparability to the original reagents for detecting and quantifying HCPs. It may also be necessary to test representative DS lots using the old vs new HCP ELISA reagents to assess the impact on DS HCP results, but only after the new reagents have been characterized for degree of comparability to the old reagents.. Even process-specific immunoreagents should be assessed for comparable sensitivity and specificity to assure continuity of data with prior reagents. An FDA case study was presented where a deficient new Pab reagent was not acceptable in assuring continued product quality and consistency for HCPs. Since an ELISA signal is a sum of all immunoreactions in the wells; the same value can come from different reactions; ie if a signal reads as100 ppm, is it from 2 x 50 ppm each, or 10 x 10 ppm each?) So ELISA alone is not capable of assessing the comparable heterogeneity of mixed populations of immunoreagents (Ag or Ab). Therefore the bridging of new HCP immunoreagents requires more than just testing the new batches in the ELISA itself. Bridging of old vs new HCP immunoreagents requires approaches that can compare the pattern and abundance of HCPs in the ELISA reference standard, and/ or the immunospecificity of the Pabs (whichever reagent was changed, or both). How should bridging studies be done between HCP assays? What are the expectations for such studies? When should they be done? Bridging the historical HCP data obtained with commercial HCP ELISA to data generated with new process-specific HCP ELISA reagents is also a critical exercise prior to finalizing HCP specifications for BLA/MAA. Typically this is done by re-testing numerous DS batches (especially all batches used in the clinic)with the new process-specific HCP ELISA to obtain more accurate values for residual HCPs. Similarly, some reported that their process clearance studies done with commercial HCP ELISAs may be re-assessed if a more specific process-specific HCP ELISA is developed.
How does one determine the suitability of HCP assays at different stages of product lifecycle? During development, when should a change be made from generic commercially available assay to the specific HCP assay? At a minimum, use an approach like 2D gels/blots to assess the specificity to your HCP proteome of the commercial ELISA immunosreagents (or immunoreagents from an existing in-house HCP ELISA ) as soon as you start using it to know what you won t know from using that ELISA. One attendee also mentioned cases of commercial HCP ELISA reagents that cross reacted with some APIs but not others in their program, confirmed by doing 1D gel/blots (reduced and non-reduced, native and denatured). It was discussed that it is wise to assess crossreaction to API before using any HCP ELISA. Hindsight has showed using process-specific ELISA as early as possible is best means to de-risk process development R&D studies missing several HCPs. You can only truly optimize the DS process steps with accurate measurement of HCPs, otherwise you will be making changes with partial data on total HCP shifts within the manfuacturing process steps.. Discussions brought up case studies that have shown inaccurate characterization of HCPs in process clearance steps can have impact on timelines if suddenly finding something not previously detected that is problematic to product and requires another round of process improvements. If using an interim HCP ELISA, it is critical to archive all DS samples tested with it for retesting with final HCP ELISA. Depending on what is detected (or not) by the early phase HCP ELISA, you may be able to use throughout development, particularly if the ELISA is supplemented with other reagent-independent HCP analytical tools in process and product characterization and comparability studies. What HCP comparability studies should be done to support process changes? 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). In process comparability studies, best practices are to balance risk of missing HCPs by ELISA by utilizing the analytical power of broad-based reagent -ndependent characterization of process and product (eg MS, 2DIGE)
Discussion points on the state-of-the art analytical control strategy for HCP: Are immunoassays still the most suitable method for QC testing of HCPs? US, HC and EU regulators answers: Currently HCP ELISAs are the gold standard for QC testing of DS for release. ELISAs generate a summed HCP response for the samples so you can t see which HCPs are there or not, but it provides a simple method for QC operations and a single value for specification limits. How to overcome dilution-related linearity issues in immunoassays with broad HCP coverage? This is the most technically challenging aspect of using highly diverse set of Pabs in a single ELISA well. (See Marty V s excellent cartoon). This is seen as hook effect in assay dilution series where lower dilutions give higher readings. HCP ELISA developers need to experimentally evaluate this phenomenon for each assay and validate the minimal valid dilution (refer to the USP HCP chapter explanation of how to do MVD). What other technologies are being used for GMP release testing for HCPs? US, HC and EU regulators answered any technology is possible if sponsors can demonstrate it accurately and sensitively measures HCPs and can be validated for reliable performance. MS is very information rich tool on HCPs (can see specific proteins and directly identify them), but may be too complex of technology for QC; Other technologies are mostly used for characterization of the QC assay as well as investigation of issues with the product/process.; some may evolve into QC uses over time. Under what circumstances is more than one QC release assay needed to measure HCPs? This could be necessary for specified HCPs like PLBL2 that are known to co-purify with DS, or other HCP sub-populatons that are residual at high abundance in DS (eg highly abundant milk proteins in DS from transgenic animals that swamp out the HCP signal from others that are at lower levels). Although in such cases sponsors might develop sufficient process characterizaton data to monitor 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 MS will also become valuable in comparability of originator to biosimilar products, although preliminary data have shown promise in assessing patterns of HCPs in biosimilar products vs originator products (Health Canada Yves Aubin)
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 those batches have been in patients (eg with a more specific HCP ELISA) is less risky than finding new HCPs in batches that have never been in clinical trials because the HCPs were already shown to be no short-term safety risk (although long term immunogenicity is unknown). For commercial products, HCP populations must be thoroughly compared before and after DS process changes to avoid surprises in the new process DS lots..for comp studies, reagent-independent techniques can be most powerful esp for comparing HCPs in process intermediates. What is the regulators thinking about routinely monitoring anti-hcps antibodies during clinical development? CBER: For replacement therapies for plasma proteins (high concentration and frequent dosing), Office of Blood Products is currently telling sponsors to develop clinical immunogenicity assay for screening anti-hcp Abs at Phase 1. Testing patients for anti-hcp antibodies (as well as the major focus of anti-drug antibodies, ADA). This is in parallel with the using an HCP ELISA for product QC testing (commercial in early phase, custom in late phase). CDER: ADA immunogenicity assay needed by ph 1 but would require anti-hcp assay on case by case basis; the risk assessment would be based on the principles of the draft FDA guidance on immunogencity of therapeutic proteins HCP spec is finalized after process validation based on clinical ranges and demonstrated process consistency. In some cases, routine HCP testing of each DS lot might be waived after commercial manufacturing consistency (eg10 to 30 lots commercial product) What are the regulators current thoughts on establishng a commonly acceptable HCP limit in biotech/biosimilar products? There are NO common HCP limits that can be officially set for all products due to multiple factors that are unique for each product: Primarily it is based on target indication for product (dose, route) and clinical purpose (replacement vs acute). Add to that the variances in HCP test method analytical capability (specificity, sensitivity), and variable process capabilities (more vs less robust clearance steps). Finally, not all HCPs are equally problematic: speaker examples showed where one specific protein caused problems in some products, while some cases rather high levels of HCPs have not been a problem in many legacy protein products. For these same reasons, with biosimiilars it is not possible to simply compare HCPs to HCP levels in similar products on the market. CBER: wide range of limits (low ng to low ug) in different units (ppm, ng/mg, ng/u) so not possible to set one limit value PEI: Limit of 1 ng/mg or less has been show to be possible for Mab products; other therapeutics generally propose NMT 100 ppm in early development
Regulatory authorities HCP assays are currently the weakest analytical link in the total characterization and control strategy for biotech and biosimilar products Excellent presentations by US and EU regulatory agencies for therapeutic and biologic products in both regions. All reg speakers listed the specific rules and guidance docs with which they are obligated to review HCPs. Please be sure all of these reg refs are in the final summary paper. Also please confirm that all reg slides are on the CaSSS website archives to accompany the paper (and point readers to these slides with the URL). 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? Speakers from academia and industry gave excellent examples of a wide variety of HCP issues observed with different types of products. (refer to speaker slides to be posted on CaSSS website for specific details). Also note that in many speaker slides were specific citations to papers on the work being presented, or similar HCP issues previously published. Review speaker slides for these useful reference articles. Along with the case studies presented, attendees (industry and regulatory) had many anecdotal candidates, but no combined list of these is available publically (yet:..?) PLBL2 in CHO (soon to be published); others have been published or presented HCP proteases (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 Request to CaSSS: Can the co-leads and speakers pull together all of the HCP case studies shown (and cited) by the presenters (esp regulators) and present the list in a side-bar table in the Forum white paper for readers to see? And where publications are available for them, please include the citations in the summary paper.