Ultrafast and high-throughput N-glycan analysis for monoclonal antibodies

Abstract

(2016) Ultrafast and high-throughput N-glycan analysis for monoclonal antibodies, mAbs, 8:4, 706-717, DOI: 10.1080/19420862.2016 To link to this article: https://doi.org/10. 1080/19420862.2016 ABSTRACTGlycosylation is a critical attribute for development and manufacturing of therapeutic monoclonal antibodies (mAbs) in the pharmaceutical industry. Conventional antibody glycan analysis is usually achieved by the 2-aminobenzamide (2-AB) hydrophilic interaction liquid chromatography (HILIC) method following the release of glycans. Although this method produces satisfactory results, it has limited use for screening a large number of samples because it requires expensive reagents and takes several hours or even days for the sample preparation. A simple and rapid glycan analysis method was not available. To overcome these constraints, we developed and compared 2 ultrafast methods for antibody glycan analysis (UMAG) that involve the rapid generation and purification of glycopeptides in either organic solvent or aqueous buffer followed by label-free quantification using matrix-assisted laser desorption/ ionization-time of flight mass spectrometry. Both methods quickly yield N-glycan profiles of test antibodies similar to those obtained by the 2-AB HILIC-HPLC method. In addition, the UMAG method performed in aqueous buffer has a shorter assay time of less than 15 min, and enables high throughput analysis in 96-well PCR plates with minimal sample handling. This method, the fastest, and simplest as reported thus far, has been evaluated for glycoprofiling of mAbs expressed under various cell culture conditions, as well as for the evaluation of antibody culture clones and various production batches. Importantly the method sensitively captured changes in glycoprofiles detected by traditional 2-AB HILIC-HPLC or HILIC-UPLC. The simplicity, high speed, and low cost of this method may facilitate basic research and process development for novel mAbs and biosimilar products.

Simultaneous monitoring of oxidation, deamidation, isomerization, and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion

“…Here, we describe an ultrafast and simple multi-attribute uLC-MS method that allows simultaneous monitoring of glycosylation, oxidation, deamidation, isomerization, glycation, and N-terminal pyroglutamate, or any modifications detectable by MS. This method adopted an ultrafast trypsin digestion protocol we developed for assays for glycan analysis 44 and site-specific oxidation, 45 and avoided the assay artifacts of oxidation, deamidation, and isomerization induced during the cLC-MS method. 8,[34][35][36][37]39 With uLC-MS available for comparison, assay artifacts for Asn deamidation and Asp isomerization at certain sites were clearly shown in the longer digestion procedure.…”

“…To increase the throughput and minimize assay artifacts, we developed uLC-MS by using an easy and fast digestion strategy that was employed for fast glycan profiling and for mAb oxidation analysis. 44,45 In this method, mAbs are first denatured and reduced in 4-8 M urea/10 mM DTT bicarbonate buffer at 70 C for 3 min, and then directly digested by trypsin at 37 C for 5 min. The alkylation and buffer exchange steps used in cLC-MS were eliminated.…”

“…44,45 Fifty mg of diluted mAb sample in 1 ml was thoroughly mixed with 20 mL of the reduction buffer (8 M urea in 20 mM ammonium bicarbonate and 10 mM DTT, and denatured and reduced at 70 C for 3 min in a water bath. Subsequently, the denatured and reduced sample was diluted 5X using 20 mM ammonium bicarbonate, and trypsin was added at a 1:20 enzyme: substrate ratio.…”

Novel chemical degradation pathways of proteins mediated by tryptophan oxidation: tryptophan side chain fragmentation

“…At this point, the exact role of glycan structure in the transfer of 3‐MEI from Trp 313 to Lys 317 is not clear; the glycan structure may affect protein conformation and flexibility, controlling the interaction of the Lys 317 side chain with the nascent 3‐MEI being released from Trp 313 . Considering the importance of glycan diversity for biosimilar development, reactions such as the formation and transfer of 3‐MEI may be useful for biosimilar analysis …”

Scale-down of CHO cell fed-batch cultures

“…Miniature chromatography columns and antibody purification platforms have also been developed that can purify mAbs at a few milligrams level 192,[236][237][238] . Several methods have enabled glycosylation analysis directly from culture supernatant and with a volume requirement in µ L ranges [239][240][241][242] .…”