Development of an LC-MS/MS peptide mapping protocol for the NISTmAb

Abstract

Peptide mapping is a component of the analytical toolbox used within the biopharmaceutical industry to aid in the identity confirmation of a protein therapeutic and to monitor degradative events such as oxidation or deamidation. These methods offer the advantage of providing site-specific information regarding post-translational and chemical modifications that may arise during production, processing or storage. A number of such variations may also be induced by the sample preparation methods themselves which may confound the ability to accurately evaluate the true modification levels. One important focus when developing a peptide mapping method should therefore be the use of sample preparation conditions that will minimize the degree of artificial modifications induced. Unfortunately, the conditions that are amenable to effective reduction, alkylation and digestion are often the same conditions that promote unwanted modifications. Here we describe the optimization of a tryptic digestion protocol used for peptide mapping of the NISTmAb IgG1κ which addresses the challenge of balancing maximum digestion efficiency with minimum artificial modifications. The parameters on which we focused include buffer concentration, digestion time and temperature, as well as the source and type of trypsin (recombinant vs. pancreatic; bovine vs porcine) used. Using the optimized protocol we generated a peptide map of the NISTmAb which allowed us to confirm its identity at the level of primary structure. Graphical abstractPeptide map of the NISTmAb RM 8671 monoclonal antibody. Tryptic digestion was performed using an optimized protocol and followed by LC-UV-MS analysis. The trace represents the total ion chromatogram. Each peak was mapped to peptides identified using mass spectrometry data. Electronic supplementary materialThe online version of this article (10.1007/s00216-018-0848-6) contains supplementary material, which is available to authorized users.

The NISTmAb Reference Material 8671 value assignment, homogeneity, and stability

“…Instrument qualification and system suitability controls were required to pass method performance criteria as outlined during method qualification [ 11 , 12 ]. Data analysis for each method was performed as described in the requisite qualification paper [ 9 , 11 , 12 ]. Combined standard uncertainty ( u c ) for RM 8671 samples were calculated as described in ESM.…”

“…The most immediate utility of the NISTmAb is for physicochemical/biophysical analysis technology development, method control, and harmonization of best practices. An expansive and continuously growing historical characterization dataset has been compiled as a foundation for these intended uses [ 3 – 8 ], a subset of which has been refined and qualified for quality monitoring [ 9 – 12 ], and used herein for quality attribute value assignment as part of the NISTmAb lifecycle management plan.…”

“…Property values may be assigned to a given RM through a variety of measurement modes, the results of which influence the value designation (certified, reference, or informational as described in) [ 10 , 13 ]. A series of method-specific protocols, each of which has been recognized by the community as common measurement methods for mAbs, were qualified to be suitable for NISTmAb measurement in the preceding papers of this series using the in-house NISTmAb primary sample (PS 8670) [ 9 – 12 ]. The physicochemical property values were then assigned to each individual lot of RM 8671 using the qualified methods and a stratified sampling and analysis plan, thereby allowing simultaneous evaluation of intra-lot homogeneity.…”

Heterologous recombinant expression of non-originator NISTmAb

“…The amino acid sequence of the NISTmAb was verified and reported using LC-MS/MS peptide mapping 10,11 . The DNA construct encoded in the cell lines developed herein was constructed to directly encode the same primary amino acid sequence, although the specific codons used for the NISTmAb were unknown.…”

Fully Automated Peptide Mapping Protocol for Multi-Attribute Method by Liquid Chromatography–Tandem Mass Spectroscopy with a High-Throughput Robotic Liquid Handling System

“…The typical protocol for preparing samples for MAM calls for denaturation, reduction, and alkylation, followed by tryptic digestion [24,25,20]. The generated tryptic peptides, which carry the product quality attributes, are then chromatographically separated and analyzed by MS. 4 Major challenges to the use of MAM, however, lie in the variability of the sample preparation procedure and analytical instruments [26,19], which can give rise to inconsistent results.…”