Sample preparation method for accurate analysis of non-enzymatic PTMs in biotherapeutic proteins with peptide mapping Sergei Saveliev, Chris Hosfield, Mike Rosenblatt and Marjeta Urh Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711 4. Establishing efficient reduction and alkylation at 6. Suppression of protein oxidation with an oxygen 1. Introduction low pH scavenger reagent Non-enzymatic chemical modifications such as deamidation, Conventional reagents used to reduce and alkylate disulfide bonds favor alkaline Various excipients and impurities can cause protein oxidation during sample preparation. disulfide bond scrambling and oxidation can affect the stability conditions. To achieve efficient reduction and alkylation at low pH we took the following To protect proteins from oxidation we selected a reagent with high oxygen scavenging steps: activity (‘oxidation suppressant’). and efficacy of biotherapeutic proteins. Peptide mapping, the . Tris(2-carboxyethyl)phosphine) (TCEP), a reducing reagent retaining high activity over a method of choice for monitoring these modifications, has a Suppression of H2O2-induced methionine oxidation with the oxidation suppressant during IgG broad pH range including mildly acidic conditions, was selected for the method. significant drawback which is that peptide mapping sample digestion (BPC) Courtesy of Quality . Iodoacetamide (IAM) was selected as an alkylating agent. To compensate for its Assistance, Belgium preparation induces the same modifications. decreased activity at acidic pH, we allowed alkylation to proceed over digestion period. W/o oxidation suppressant We developed a sample preparation method which prevented the above side effects. In our method, all sample preparation Counts) steps including reduction, alkylation and digestion are performed ( BPI at low pH (mildly acidic conditions). Efficient reduction and Procedure for protein reduction MS analysis of Panitumumab reduced and alkylated With oxidation suppressant alkylation at low pH utilized both specialized reagents and and alkylation at low pH according to the low pH method procedural modifications. The proteolytic step represented the Alkylated peptides Peptide peak intensity Retention time, min Reduction with TCEP, major bottleneck since trypsin, the most commonly used A model IgG (Adalimumab) was denatured, reduced, alkylated and digested according to the low pH 37oC 30 min Alkylated Non-alkylated sample preparation procedure in the presence of an oxidation agent (hydrogen peroxide) with or protease in peptide mapping, is inhibited at acidic pH. We CCVECPPCPAPPVAGPSVFLFPPKPK 1.69E+10 2.05E+07 without the oxidation suppressant. The digests were analyzed with LC/MS. The data indicate overcame this problem by supplementing trypsin with a low pH LSSVTAADTAIYYCVR 6.4E+10 8.66E+07 suppression of oxidation. resistant Lys-C protease. We also selected a reagent with oxygen NQVSLTCLVK 1.66E+11 1.75E+06 Alkylation with IAM, Suppression of H O -induced methionine oxidation during IgG digestion scavenging activity, which was successfully applied to suppress 37oC 30 min (initial alkylation step) SGTASVVCLLNNFYPR 1.86E+11 6.93E+06 2 2 (oxidized methionine residues, %) Courtesy of Quality protein oxidation during sample preparation. Cysteines were alkylated at >99% efficiency. Assistance, Belgium Panitumumab was reduced with TCEP, alkylated with IAM, The method was optimized for use with common sample Digestion (alkylation is allowed to digested and analyzed with LC/MS (Q Exactive, Thermo). 80% preparation procedures based on protein denaturation followed proceed over digestion period) All steps were performed at low pH as shown on the left. Oxidation in the absence of by dilution or size exclusion clean-up. Necessary optimizations Note: Iodoacetic acid (IAA) supported similar efficient the oxidation suppressant alkylation in our low pH method. 50% were introduced to ensure efficient digestion of proteolytically- Oxidation in the presence of resistant protein domains, minimal baseline noise in UV HPLC the oxidation suppressant and LC/MS and high analytical reproducibility. HC:M34ox HC:M83ox HC:M256ox HC:M432ox LC:M4ox Efficient reduction and alkylation was achieved at low pH requiring minimal procedural The oxidation suppressant suppressed protein oxidation during sample preparation in a modifications. model experiment. 2. Artificial non-enzymatic PTMs induced during 5. Adaptation of the method for common sample 7. Case study: BioSimilar vs Originator comparison sample preparation and low pH solution preparation procedures Asn deamidation and Asp isomerization induced during protein digestion The low pH sample preparation method was applied to investigate structural similarity Peptide mapping sample preparation at low pH between BioSimilar (Remsima) and Originator (Remicade). Courtesy of Alexia Ortiz and Koen Sandra (RIC, Belgium) Protein denaturation in a concentrated GuHCl or Urea reconstituted in a low pH buffer Collaborative study by Anna Schwendeman et al. (U.Michigan), MS BioWorks, LLC; Protein Metrics Inc. and Promega Corporation. Peptide 1 Asn deamidation Peptide 3 Asp isomerization Sample preparation under non-reducing conditions Conventional Conventional Reduction with TCEP at low pH. Deamidation in Remicade and Remsima digestion (pH8) digestion (pH8) Sample preparation 37oC 30 min Deamidation was overestimated is complete within 5h. Blocking unpaired cysteines with Deamidation, % Deamidation, if IgGs were prepared according Low pH digestion % Isomerization, Low pH digestion N-Ethylamaleimide (NEM) at low pH, to conventional procedure. Low Alkylation with IAM at low pH. 37oC 30 min 37oC 30 min (initial alkylation) pH procedure showed very low if Digestion, h Digestion, h any deamidation in Remicade mAb was digested at 37oC at conventional (pH8) or low pH (mildly acidic) conditions. Aliquots of the and Remsima. digests were collected over the course of digestion reaction and analyzed with LC/MS. % Deamidation, Dilution-based procedure Desalting-based procedure Disulfide bond scrambling induced during sample preparation Deamidation sites LC194-LC214 scrambled disulfide Disulfide bond scrambling in Remicade and Remsima bond peptide pair Pre-digestion of tightly folded protein Desalting with size exclusion Disulfide bond scrambling was increased in domains at strong denaturing conditions chromatography Remsima in comparison to Remicade if sample preparation was performed under conventional XIC analysis and low pH with low pH resistant Lys-C. 1h 37oC conditions (scrambling induction was, probably, caused by an excipient in Remsima). This Scrambled bond Peak intensity, % intensity, Peak discovery proved to be false as both IgGs showed Dilution to 0.5-1M GuHCl or Digestion with trypsin and low pH virtual lack of scrambling if sample preparation 2-2.5M Urea with low pH buffer resistant Lys-C or low pH resistant was performed under low pH conditions. pH8 digest Lys-C alone at low pH. 3h 37oC Scrambled disulfide bonds Completion of digestion with trypsin Remicade, conventional sample prep (pH8) Remsima, conventional sample prep (pH8) Scrambling is and low pH resistant Lys-C or low pH suppressed. Low pH digest resistant Lys-C alone at low pH. Remicade, low pH sample prep Remsima, low pH sample prep pH8 digest Low pH digest 3h 37oC IgG was digested according to conventional (pH8) or low pH procedure and analyzed with LC/MS o Panitumumab was digested at 37 C at conventional (pH8) or low pH (mildly acidic) conditions and (Q Exactive, Thermo). disulfide bond scrambling was analyzed with LC/MS. Optimization of protein digestion at low pH (dilution-based procedure with GuHCl as a denaturing agent), RP HPLC analysis of Panitumumab digests Alkaline conditions favored by conventional sample preparation induce non-enzymatic Low pH sample preparation provided the means for accurate comparison of BioSimilar o PTMs. Suppression of these PTMs requires use of acidic conditions (low pH). 1:1:50 trypsin:Lys-C:protein ratio; 37 C overnight and Originator. (some peptides are poorly digested) Digested peptides 3. Establishing efficient trypsin digestion at 1:1:5 trypsin:Lys-C:protein ratio; 37oC overnight (baseline noise ( ) is induced due to overdigestion) 8. Conclusions low pH Digested peptides Step 1. Determining the reaction pH for suppression of artificial PTMs 1:1:5 trypsin:Lys-C:protein ratio; 37oC 3h (optimal) . We have developed a new peptide mapping method in which all sample Monitoring of deamidation during Panitumumab was digested overnight at Digested peptides protein digestion at increasing 37oC at indicated pH and analyzed with preparation steps are performed at low pH (mildly acidic conditions). reaction pH LC/MS. The graph shows analysis of SGTASVVCLLNNFVPR tryptic peptide used . We achieved robust tryptic protein digestion at low pH by Selected pH range for low as a deamidation reporter. supplementing trypsin with with a specialized, low pH resistant Lys-C. pH sample preparation Side-by-side comparison of alternative digestion procedures, Deamidation was induced at above pH6. Deamidation, % Deamidation, RP HPLC analysis of Panitumumab digests We selected pH5.7-5.8 as an optimal . The method was optimized for efficient reduction, alkylation and pH5 pH5.5 pH5.7 pH5.8 pH6.0 pH6.4 pH8.0 reaction pH to suppress artificial PTMs. Conventional overnight digestion efficiency at low pH, as well as minimal baseline noise and Digested peptides digestion with trypsin (pH8) high reproducibility. It was successfully incorporated into common