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Protein Metrics Solutions Vendor Neutral Host Cell Protein Analysis Regulator Positions on HCPs – VERY CLEAR • Regulators classify residual host cell proteins (HCPs) as “process-related impurities” [ICH Q6B, Q2B]. • ‘Finding’, ‘measuring’ and ‘monitoring’ are key guideline elements • EMA from 1997: ‘The ability of the purification process to remove other specific contaminants such as host-cell proteins … should be demonstrated’ • USFDA biosimilar guidelines 2012: “In all cases, the chosen analytical procedures should be adequate to detect, identify, and accurately quantify biologically significant levels of impurities (see ICH Q2B)”. http://www.fda.gov/downloads/drugs/guidancecomplianceregula http://www.ema.europa.eu/docs/en_GB/document_librar toryinformation/guidances/ucm073488.pdf y/Scientific_guideline/2009/09/WC500003947.pdf Before there was Mass Spectrometry… Points at which Cost Savings Can be Made with HCP Analysis Upstream: ‘Catalogue’ of HCPs/ contaminants – Save Millions of dollars over product lifecycle and multiple cell lines Image source: Ratcliff and Preisig 2013; BioProcess International; http://www.bioprocessintl.com/journal/2013/April/Ad vances-in-Sensor-Technology-Improve- Biopharmaceutical-Development-341848 Points at which Cost Savings Can be Made with HCP Analysis Upstream: ‘Catalogue’ of HCPs/ contaminants – Save Millions of dollars over product lifecycle and multiple cell lines Improved ELISA assays (better specificity) – hundreds of thousands of dollars in avoided recall per batch Image source: Ratcliff and Preisig 2013; BioProcess International; http://www.bioprocessintl.com/journal/2013/April/Ad vances-in-Sensor-Technology-Improve- Biopharmaceutical-Development-341848 Points at which Cost Savings Can be Made with HCP Analysis Upstream: ‘Catalogue’ of HCPs/ contaminants – Save Millions of dollars over product lifecycle and multiple cell lines Improved ELISA assays (better specificity) – hundreds of thousands of dollars in avoided recall per batch Time to market: days of method development vs months for raising polyclonals (200k+) Image source: Ratcliff and Preisig 2013; BioProcess International; http://www.bioprocessintl.com/journal/2013/April/Ad vances-in-Sensor-Technology-Improve- Biopharmaceutical-Development-341848 Points at which Cost Savings Can be Made with HCP Analysis Upstream: ‘Catalogue’ of HCPs/ contaminants – Save Millions of dollars over product lifecycle and multiple cell lines Improved ELISA assays (better specificity) – hundreds of thousands of dollars in avoided recall per batch Time to market: days of method development vs months for raising polyclonals (200k+) Process development removal of HCPs – Tens of thousands of dollars per purification Image source: Ratcliff and Preisig 2013; BioProcess International; http://www.bioprocessintl.com/journal/2013/April/Ad vances-in-Sensor-Technology-Improve- Biopharmaceutical-Development-341848 The problem with Animal Models – not HUMAN • Immunogenicity to HCPs varies by organism. Serum raised in animal models cannot always predict immunogenicity in humans. http://www.ema.europa.eu/docs/en_GB/document_li brary/Scientific_guideline/2009/09/WC500003947.pdf EMA : “The predictive value of animal models for evaluation of immunogenicity is low due to inevitable immunogenicity of human proteins in animals”. The problem with Animal Models – not HUMAN • Immunogenicity to HCPs varies by organism. Serum raised in animal models cannot always predict immunogenicity in humans. http://www.ema.europa.eu/docs/en_GB/document_li brary/Scientific_guideline/2009/09/WC500003947.pdf EMA : “The predictive value of animal models for evaluation of immunogenicity is low due to inevitable immunogenicity of human proteins in animals”. ELISA vs Mass Spec for HCPs • Which technique is ‘best’? Neither: both techniques offer some advantages, although LCMSMS has the potential for a better ROI when considering RISK, TIME and General Applicability. Attribute ELISA LCMSMS Notes Specificity LOW HIGH LCMSMS is blind to protein type; identifies individual proteins (assuming a dB) Cost Low at point of High Capital LCMSMS can save TIME – and is generic. ELISAs tend to be process-specific, although use; costly to cost; cost per are cheap to use once developed. develop sample low Deployment Simple Need a level of ELISAs can be used by almost anyone with a minimum of training; MS techniques expertise require at least a small amount of skill/ knowledge Regulators Well established / Increasing NO OFFICIAL GUIDELINES about TECHNIQUES for HCPs: regulations provide guidelines understood acceptance that ask the developer to show the most appropriate technology for the task. Therefore it will depend on the cell line, the process, the therapeutic etc. Applicability Process-specific Widely WCBP 2015 and other posters : time taken to analyse multiple cell lines with generic applicable methods in weeks; ELISAs generally take >8 months. Immunogenicity Animal models No animals MS is ‘blind’ to which protein is immunogenic, providing an unbiased assessment; harmed ELISAs developed in animal models may not reflect HUMAN reactions, and therefore this is an unknown risk. NB: LCMS is not an assay for immunogenicity ‘Absolute’ Very HIGH [ppb] Low ppm ELISAs that are well developed can be exceptionally sensitive, to the ppb level. sensitivity However, over a wide dynamic range MS can provide a good statistical set. BUT: no amount of sensitivity helps if you miss a potentially dangerous protein! ELISA vs Mass Spec for HCPs • Which technique is ‘best’? Neither: both techniques offer some advantages, although LCMSMS has the potential for a better ROI when considering RISK, TIME and General Applicability. Attribute ELISA LCMSMS Notes Specificity LOW HIGH LCMSMS is blind to protein type; identifies individual proteins (assuming a dB) Cost Low at point of High Capital LCMSMS can save TIME – and is generic. ELISAs tend to be process-specific, although use; costly to cost; cost per are cheap to use once developed. develop sample low Deployment Simple Need a level of ELISAs can be used by almost anyone with a minimum of training; MS techniques expertise require at least a small amount of skill/ knowledge Regulators Well established / Increasing NO OFFICIAL GUIDELINES about TECHNIQUES for HCPs: regulations provide guidelines understood acceptance that ask the developer to show the most appropriate technology for the task. Therefore it will depend on the cell line, the process, the therapeutic etc. Applicability Process-specific Widely WCBP 2015 and other posters : time taken to analyse multiple cell lines with generic applicable methods in weeks; ELISAs generally take >8 months. Immunogenicity Animal models No animals MS is ‘blind’ to which protein is immunogenic, providing an unbiased assessment; harmed ELISAs developed in animal models may not reflect HUMAN reactions, and therefore this is an unknown risk. NB: LCMS is not an assay for immunogenicity ‘Absolute’ Very HIGH [ppb] Low ppm ELISAs that are well developed can be exceptionally sensitive, to the ppb level. sensitivity However, over a wide dynamic range MS can provide a good statistical set. BUT: no amount of sensitivity helps if you miss a potentially dangerous protein! How Do You Explain Your Product to the FDA? How Do You Explain Your Product to the FDA? In its constituent parts… The Push to Higher Titer has Increased Challenges in Purification • Cell lines are being increasingly ‘pushed’ or ‘stressed’ Source: Bioprocess International, June 2009 The Push to Higher Titer has Increased Challenges in Purification • Cell lines are being increasingly ‘pushed’ or ‘stressed’ • Purification processes have become longer, more complex, and more expensive • Better understanding of the process saves costs Source: Bioprocess International, June 2009 Host Cell Proteins (HCPs): Unpredictable? Wang et al, Biotechnology and Bioengineering, Vol. 103, No. 3, June 15, 2009 • “The physiochemical properties of the intended recombinant protein … influence the HCPs present at various stages in the bioprocess due to co-purification .... These factors can be quite difficult to predict a priori and are often learned only by testing during process development.” • .. So it is wise to use a comprehensive approach Mass Spectrometry for HCPs: A bit of History… 2008 – 2016 2012 Improved Isotopically 2017 Eli Lilly study Dynamic Range labelled analog with Protein 2008 MALDI, re- 2009 - 2016 Data (2D-LC, Ion peptides Metrics Software use of Independent Mobility) 2014 CE-MS/MS Proteomics tools techniques techniques Selective; No Quantitation http://www.bioprocessintl.com/manufactu ring/monoclonal-antibodies/proteomics- technology-applied-to-upstream-and- downstream-process-development-of-a- High Analyst Skill level, More generalized approaches, protein-vaccine-182166/ Manual processing (days) wider applicability DOI 10.1002/biot.201500550 Targeted, Absolute doi: 10.4161/mabs.4.1.18748 Quantitation, low throughput http://dx.doi.org/10.1080/19420862.2017.1303023 DOI 10.1002/btpr.2530 A Competitive Space – MS Vendor Methodologies Vary Widely • All Mass Spec Vendors are showing that they can do HCPs … differently https://www.bruk er.com/fileadmin/ user_upload/8- PDF- Docs/Separations_ MassSpectrometry /Literature/Applica tionNotes/LCMS- 101_Protein_Thera peutics_02- 2015_ebook.pdf https://www.thermofisher.com/order/catalog /product/A27601 https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC3338939/ https://www.youtube.com/watch?v=q https://sciex.com/host-cell-protein-detection-without-pain-below- 0qZeTTGzOA 1ppm-for-all-universes-by-1d-ms
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