Improving the throughput of immunoaffinity purification and enzymatic digestion of therapeutic proteins using membrane-immobilized reagent technology

Analyst. 2020 Apr 14;145(8):3148-3156. doi: 10.1039/d0an00190b.

Abstract

Continued interest in protein therapeutics has motivated the development of improved bioanalytical tools to support development programs. LC-MS offers specificity, sensitivity, and multiplexing capabilities without the need for target-specific reagents, making it a valuable alternative to ligand binding assays. Immunoaffinity purification (IP) and enzymatic digestion are critical, yet extensive and time-consuming components of the "gold standard" bottom-up approach to LC-MS-based protein quantitation. In the present work, commercially available technology, based on membrane-immobilized reagents in spin column and plate format, is applied to reduce IP and digestion times from hours to minutes. For a standard monoclonal antibody, the lower limit of quantitation was 0.1 ng μL-1 compared to 0.05 ng μL-1 for the standard method. A pharmacokinetics (PK) study dosing Herceptin in rat was analyzed by both the membrane and the standard method with a total sample processing time of 4 h and 20 h, respectively. The calculated concentrations at each time point agreed within 8% between both methods, and PK values including area under the curve (AUC), half-life (T1/2), mean residence time (MRT), clearance (CL), and volume of distribution (Vdss) agreed within 6% underscoring the utility of the membrane methodology for quantitative bioanalysis workflows.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Chromatography, Affinity / methods*
  • Enzymes, Immobilized / chemistry*
  • Male
  • Membranes, Artificial*
  • Proof of Concept Study
  • Proteolysis
  • Rats, Sprague-Dawley
  • Staphylococcal Protein A / chemistry
  • Time Factors
  • Trastuzumab / analysis*
  • Trastuzumab / chemistry
  • Trastuzumab / isolation & purification
  • Trastuzumab / pharmacokinetics
  • Trypsin / chemistry

Substances

  • Enzymes, Immobilized
  • Membranes, Artificial
  • Staphylococcal Protein A
  • Trypsin
  • Trastuzumab