Can Standardized/Kitted Methods for Protein Digestion be Advantageous to the Laboratory? A monoclonal antibody example.

Monoclonal antibodies (mAbs) are becoming commonplace treatment for cancers, autoimmune diseases, and other diseases thought to be untreatable in the past. Therapeutic drug monitoring of these biologic therapies is of clinical significance, especially as some of these mAb therapeutics are lifelong treatments with great expense. Development of methods to monitor drug concentrations in serum comprises immunoassays or mass spectrometry pathways, including intact light chain quantitation or the use of tryptic peptides unique to the mAb. We have found the tryptic approach to be highly complex with sample clean-up, reduction, alkylation, and trypsin digestion. Many of these steps are manual, with long incubation times and can be prone to error in a clinical laboratory. As future mAb peptide detection methods are implemented into the clinical laboratory setting, the standardization and improved traceability of pre-analytical steps for parts or the entire assay could be advantageous.

Our goal was to perform a method comparison between a laboratory-developed manual sample preparation protocol using in-house reagents and a kitted digest method (Waters ProteinWorks Direct Digest Kit, Waters Inc.) for the measurement of infliximab. Residual waste serum samples with physician-ordered infliximab testing were obtained from the clinical laboratory (n=39). Samples were processed per standard operating procedure. Briefly, a protein level standard was added to the serum, and the mixture underwent a protein crash using saturated ammonium sulfate (SAS). The reconstituted pellet was then denatured, reduced, alkylated, and digested as previously published [1]. Reagents for this process are weighed and reconstituted daily. Side by side, the SAS mixture was prepared and the digest was performed using the kitted digest reagents, which incorporates denaturation, reduction, alkylation, and digestion steps, following manufacturer’s directions. Analysis of samples prepared by both methods was performed by LC-MS/MS on an API 5000 triple quadrupole mass spectrometer (ABSciex).

Sample processing using the standard operating procedure has a turn-around-time (TAT) >10h, from the initial denaturation to the digestion quenching. The kitted digest method can be performed within 4 hours, completing sample preparation in a single shift. The accuracy of infliximab quantitation using the kitted reagents was measured using ordinary linear regression; y=1.02x+1.10; R²=0.99. Additionally, quantitation using tryptic peptides m/z unique to the light chain or the heavy chain of infliximab yielded similar results; y=0.97x-0.21, R²=0.96, and may be used as a different measure of accuracy and complete digestion.

The kitted method is amiable to any upstream enhancement/enrichment steps that maybe employed with our mAb workflow and results in equivalent quantitation of infliximab in residual serum samples. Although precision and cost still need to be fully evaluated, this initial proof-of-concept comparison showed important advantages. These advantages include lot traceability, ready-to-use reagents and a significant improvement in TAT compared to our current method, which could allow better follow up and accommodate an eventual increase in test volumes by adding efficiency to the sample pre-analytical processes without a substantial method change.

1.            Willrich, M.A., Murray, D.L., Barnidge, D.R., Ladwig, P.M., Snyder, M.R.: Quantitation of infliximab using clonotypic peptides and selective reaction monitoring by LC-MS/MS. Int Immunopharmacol. 28, 513-520 (2015)