title: High resolution separation of recombinant monoclonal antibodies by size-exclusion ultra-high performance liquid chromatography (SE-UHPLC).
abstract: Size-exclusion chromatography (SEC) is an important mode of separation used in monoclonal antibody (mAb) characterization and quality control. SEC separates mAbs into three major species: high molecular weight species, main peak (predominantly monomer), and low molecular weight species. However, mAb SEC separations have low resolution between the different sized species, and the analysis is slow with low sample throughput. The introduction of size-exclusion ultra-high performance liquid chromatography (SE-UHPLC) columns offers a new opportunity to improve both the resolution and throughput of SEC analysis. This study demonstrates that SE-UHPLC columns deliver better resolution of size variants in a shorter period of time than conventional SEC columns. For example, an SE-UHPLC column 300-mm in length produced separation of mAb Fab/c fragments in less than 10min, in comparison to a conventional SEC column output, where these fragments co-elute with the main peak. Furthermore, we observed that high back pressure does not generate HMWS under optimized mobile phase conditions for mAbs. The platform SE-UHPLC method has been demonstrated to be suitable for the analysis of multiple mAbs, with greatly improved sample throughput and peak resolution of mAb size variants.
authors.affiliation: Department of Analytical Operation, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Renee
authors.initials: R
authors.lastname: Yang
authors.affiliation: Department of Analytical Operation, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address: yuntang@gene.com.
authors.forename: Yun
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Department of Protein Analytical Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Department of Analytical Operation, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Lu
authors.affiliation: Department of Analytical Operation, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Alice
authors.initials: A
authors.lastname: Liu
authors.affiliation: Department of Protein Analytical Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address: yonghuaz@gene.com.
authors.forename: Yonghua Taylor
authors.initials: YT
authors.lastname: Zhang
mesh_terms: D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D007140:Immunoglobulin Fab Fragments / Q000032:analysis; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012984:Software; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins
keywords: High pressure; Monoclonal antibodies; SE-UHPLC; SEC; Theoretical plates