Research Article
ELISA microplate: a viable immunocapture platform over magnetic beads for immunoaffinity-LC–MS/MS quantitation of protein therapeutics?
Aim: Evaluate the performance of ELISA microplates versus commonly used magnetic beads for biological sample cleanup and/or enrichment in immunoaffinity-LC–MS/ MS to reduce tedious beads washing procedures and a relatively high assay cost. Materials & Methods: ELISA microplates were used as immunicapture platform and compared with magnetic beads for sample cleanup for LC–MS/MS quantitation of protein therapeutics. Results: One unmodified and two surface-activated microplates provided comparable linear ranges and sensitivities for a therapeutic protein (mass 78 kDa) using a human serum sample of 100 μl with 1:1 dilution compared with Tosylactivated magnetic beads using 200 μl of human serum without sample dilution. The assays’ precision and accuracy were all within acceptable ranges. No nonspecific binding or other selectivity issues were observed. Conclusion: The results suggested an ELISA microplate could be a viable immunocapture platform for immunoaffinity- LC–MS/MS quantitation of protein therapeutics.
LC–MS/MS technology has been widely recognized as a complementary methodol- ogy to ligand-binding assay for quanti- tation of protein therapeutics [1,2]. This is driven by the growing demand from the bio-pharmaceutical industry in support of the development of protein therapeutics, and is also attributed to its promising features such as fast method development, wide linear dynamic range and high selectivity in con- trast to ligand-binding assay. While its suc- cess has been evidenced in the past decade, the approach has many challenging aspects, particularly sample preparation. Although intact peptides and smaller pro-
teins (typically MW < 10 kDa) could be directly quantitated by LC–MS/MS, enzy- matic digestion to release the surrogate peptides from the protein of interest for detection still remains the most general approach [1]. Consequently, conducting sample pretreatment prior to the digestion is desired for successful method development. This is because common biological matri- ces, such as serum or plasma, contain large amounts of endogenous proteins which nega-
10.4155/BIO.14.250 © 2015 Future Science Ltd
tively impact direct digestion of protein ther- apeutics, generally compromising the entire assay performance. A variety of sample pretreatment tech-
niques are available, unfortunately not many are applicable for the treatment of high- molecular weight protein therapeutics in bio- logical matrixes in automated format [3]. A simple improvement of direct digestion, ‘pel- let-digestion’, or proteolytic digestion of the pellet obtained following solvent-mediated co-precipitation, has achieved success. The approach used a small sample volume (25 or 50 μl), which limits the improvement of assay LLOQ. The reported LLOQs were above μg/ml level [4,5]. Protein depletion is another choice. By depleting abundant proteins, such as albumin and immunoglobulins, using dyes (chemical-based) or polyclonal antibod- ies (immunoaffinity-based), sample com- plexity could be significantly reduced. Using this approach combined with LC–MS/MS, a human mAb quantitation assay in monkey serum was reported with a linear range of 2–1000 μg/ml [6]. One drawback with this approach is the procedure might lack selec-
Bioanalysis (2015) 7(3), 307–318 ISSN 1757-6180
Wenchu Yang*,1 Kernstock1
& Ala Alak1 1
, Robert , Neal Simmons1 Bioanalysis-US, Astellas Research
Institute of America, Skokie, IL 60077, USA *Author for correspondence: Tel.: +1 847 933 7494 Fax: +1 847 933 7482
wenchu.yang@astellas.com
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