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Host cell protein analysis by mass spectrometry and its application in a comparability exercise Florian Wolschin, Martin Schiestl CMC Strategy Forum, Washington, 26. Jan 2015 Topics Introduction Use of mass spectrometry in HCP analysis Application in a comparability exercise Role of mass spectrometry versus ELISA in HCP analysis • Comparability following a manufacturing change • Biosimilar exercise 2 | 26 Jan 2015 2 The cell Source: Animal cell. Wikimedia Commons, author: Mediran. Host Cell Proteins (HCP) including modifications 3 | 26 Jan 2015 HCP immuno assay development Standard approach Cell line without product-coding gene Immunization Bioprocess Purification and qualification of polyclonal antibody sera Isolation Purification steps 4 | 26 Jan 2015 Immunoassay development Choose suitable step to take HCP preparation ELISA and related assay formats LC-MS in HCP analysis Mass spectrometry allows the identification of low abundant proteins in complex mixtures • Increasing use of LC-MS techniques in HCP analysis • Ongoing progress in increasing sensitivity and mass accuracy LTQ OrbitrapTM 5 | 26 Jan 2015 Q-ExactiveTM Analysis of proteins in complex mixtures Database search concept Protein Protein Digestion 6 | 26 Jan 2015 HCP LCMS identification workflow Process sample Sample Drug substance Capture eluate (Protein A purified) Protein digestion (e.g. Trypsin) LC-MS/MS Database search of MS/MS spectra + Exclusion list Derived from API peptides + Inclusion list Derived from HCPs identified in capture eluate Library of potential HCPs In drug substance 7 | 26 Jan 2015 7 Effect of using exclusion and inclusion lists Increase of detectability 8 | 26 Jan 2015 How reliable are the identifications? MS/MS spectra verification with synthetic peptides 9 | 26 Jan 2015 V. Reisinger, H. Toll, R.E. Mayer, J. Visser, F. Wolschin, Analytical Biochemistry 463 (2014) 1-6. HCP analysis in a comparability exercise Case: Exchange of the depth filter in the manufacturing process ELISA results showed comparable levels of HCP in the drug substance batches before and after the filter change LC-MS/MS using inclusion and exclusion lists • Pre- and post change batches were analyzed for HCPs using automatic identification • In addition, all HCPs automatically identified in one of the two sample sets were manually checked for their presence in the other 10 | 26 Jan 2015 V. Reisinger, H. Toll, R.E. Mayer, J. Visser, F. Wolschin, Analytical Biochemistry 463 (2014) 1-6. 10 HCP analysis in a comparability exercise Protein Identified before filter change Identified after filter change Protein S100-A10 X X Protein S100-A4 X X Protein S100-A11 X (X) Protein S100-A6 X (X) Anionic trypsin-2 X (X) Thioredoxin X X Galectin-1 X (X) X ... Identified by automatic analysis (X) ... Identified by manual analysis 11 | 26 Jan 2015 V. Reisinger, H. Toll, R.E. Mayer, J. Visser, F. Wolschin, Analytical Biochemistry 463 (2014) 1-6. 11 Arbitrary units Arbitrary units Exemplary HCP quantification results 12 | 26 Jan 2015 Batches before filter change Batches after filter change Quantification workflow LC-MS/MS with accurate mass (Q-Exactive) Semi-automated data evaluation for identification and quantification. De novo sequencing possible. Manual verification of identification and integration Quantification of peptides using targeted MS/MS 13 | 26 Jan 2015 Manual versus automated quantitative analysis Standard protein spicked into DS 25000000 Manual y = 51790x - 210171 R² = 0.982 7000000 y = 61110x - 687950 R² = 0.9775 6000000 Arbitrary units 20000000 5000000 15000000 4000000 3000000 10000000 2000000 5000000 1000000 0 0 0 100 200 300 400 500 -1000000 [ng/mg] 0 20 y = 7110.9x + 50667 R² = 0.9695 Arbitrary units 3500000 3000000 2500000 2000000 1500000 1000000 500000 0 -100 14 | 26 Jan 2015 0 100 200 300 400 60 80 100 120 [ng/mg] Automated 4000000 40 500 [ng/mg] Poor correlation of ELISA and quantitative MS Exemplary graphs 0.80 Pearson 0.70 Spearman HCP #13 0.60 0.50 0.40 0.30 0.20 MS Correlation value ELISA/MS 0.90 0.10 HCP #2 0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 HCPs Possible reasons • Narrow HCP distribution in process samples • Method variability • Complementary HCP recognition 15 | 26 Jan 2015 ELISA Physicochemical HCP properties Proof of principle samples Attribute Median CapE HCPs (n=48) pI Median DS HCPs (n=8) p-value 5.59 5.49 0.9862 mw [Da] 50651.88 20297.51 0.0662 GRAVY -0.25 -0.37 0.2079 Samples before filter change Attribute pI Median CapE HCPs (n=33) 5.55 mw [Da] 41991.88 11239.58 0.0009 GRAVY -0.25 -0.25 0.6564 Attribute pI Median DS HCPs (n=7) p-value 5.49 0.9931 Samples after filter change Median CapE HCPs (n=24) Median DS HCPs (n=3) p-value 5.49 5.23 0.9891 mw [Da] 41867.28 11239.58 0.0304 GRAVY -0.34 -0.33 0.5227 16 | 26 Jan 2015 V. Reisinger, H. Toll, R.E. Mayer, J. Visser, F. Wolschin, Analytical Biochemistry 463 (2014) 1-6. Role of MS versus ELISA ELISA is the assay of choice for routine analysis • Precise and sensitive assay format • Provides relative but no absolute quantitative results • Due to HCP-specific immunogenicity some HCPs may react stronger than others • Despite some limitations, history demonstrated the appropriateness of HCP control by well developed ELISAs MS complements ELISA • Identification and quantification of single HCPs • Capture more abundant HCPs in process samples and drug substance 17 | 26 Jan 2015 17 Role of MS versus ELISA Comparability exercises following manufacturing process changes • ELISA is the routine tool to compare HCP clearance before and after the change • MS provides characterization data for the more abundant HCPs – Useful addition for risk mitigation or trouble shooting Biosimilar exercises • HCP is a process related feature • A biosimilar company needs to justify adequate low levels of HCPs for their own process • – Different processes may result in different HCPs – Therefore, a direct comparison with the reference product is normally not useful HCP-ELISA needs to be qualified for a specific process and is not suitable to compare HCPs load of products resulting from differently developed manufacturing processes 18 | 26 Jan 2015 18 HCP content HCP content HCP content HCP content Biosimilar exercise: HCP-ELISAs are not suitable to compare different manufacturing processes 19 | 26 Jan 2015 19 Summary Mass spectrometry is a useful tool for HCP analysis MS-based label free methods are capable of identifying and quantifying individual proteins down to single digit ppm (ng/mg) Exclusion and inclusion lists can enhance the sensitivity of HCP detection • Be aware of potential bias when using inclusion lists Mass spectrometry provides complementary results to ELISA • Identification and quantitation of (more abundant) HCP proteins – • May compensate limitations of ELISA Useful in evaluating manufacturing process changes Biosimilar exercise • As a process related feature, HCP comparison is normally not useful • HCP-ELISA needs to be qualified for a specific process and is not suitable to compare products from differently developed processes 20 | 26 Jan 2015 All trademarks shown in this slide deck are the property of their respective owners. 20