MSACL 2023 Abstract

Introduction:
Bottom-up protein quantification requires digestion of the target protein into component peptides. The abundance of selected peptides is accepted to be a surrogate for the abundance of the protein. However, this method requires that proteotypic peptides be unique and not found endogenously in the target matrix. Adalimumab (Humira) is a monoclonal antibody (mAb) tumor necrosis factor (TNF) inhibitor that is widely used to treat a variety of inflammatory conditions, such as rheumatoid arthritis, Crohn’s disease, and ulcerative colitis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for mAb quantification in complex matrices are desirable because they allow for measurement of total drug rather than free, are generally more accurate and specific than antibody-based methods, and allow for assay multiplexing. The use of LC-MS/MS internal standards (ISTDs) improves analytical variation by correcting bias and variation. Immunoanalysis methods do not use ISTDs and therefore are prone to variability due to reagents, biologic interferences, etc. Despite literature claiming adalimumab is quantifiable using a simple bottom-up proteomics approach, due to the possibility of endogenous peptides it is likely not possible without utilizing immunoenrichment to select for adalimumab. Adalimumab is a fully-humanized mAb, which reduces its immunogenicity, increases its half-life, and allows for human effector functions to take place at Fc receptors. However, unlike chimeric mAbs which may contain multiple species-specific peptides unlikely to be found in humans, adalimumab did not contain any unique peptide sequences.

Methods:
The adalimumab protein sequence was subjected to in silico tryptic digestion using PeptideMass. Using the peptide sequences generated in silico and with agreement from literature, the peptides APYTFGQGTK (light chain) and NYLAWYQQKPGK (light chain) were selected for evaluation. Standard peptides were purchased from GenScript. Sample preparation consisted of protein enrichment with saturated ammonium sulfate, resuspension of the pellet in tris buffer, denaturation with trifluoroethanol, reduction with dithiothreitol, and alkylation with iodoacetamide. The protein was then digested with trypsin overnight and analyzed using a reversed-phase chromatography method with a Zorbax RR Eclipse XDB-C8 trapping cartridge (2.1 mm x 15 mm, 3.5 µM), an Acquity UPLC Peptide CSH C18 VanGuard Pre-column (2.1 mm x 5 mm 1.7 µM, 130 Å), and an Acquity UPLC Peptide CSH C18 Column (2.1 mm x 100 mm, 1.7 µM, 130 Å) on a Thermo Fisher Scientific Altis MS/MS coupled to a Thermo Fisher Scientific UltiMate3000 Dionex LC.

Results:
When ten naïve patient serum samples were screened for adalimumab, peaks were observed for APYT in eight samples and NYLA in nine samples. The peaks were all above the limit of quantitation of 1 µg/mL. The interferences were likely due to endogenous peptides as the interference signals increased with increased sample volume. Additionally, the interferences were not observed in various blank samples/solvents. Upon further research using UniProt, evidence of both APYT and NYLA was found at the transcript level in human Ig protein.

Conclusion:
Adalimumab was not able to be quantified via LC-MS/MS using the desired sample preparation technique due to the lack of multiple identifiable unique tryptic peptides. Despite the typically high specificity of LC-MS/MS methods, in this case antibody-based quantification methods are more specific and alternative methodologies must be explored. If LC-MS/MS analysis was required, a more complicated sample preparation could remediate the problem, e.g. affinity resin. However, at higher cost and number of sample preparation steps.