title: Inline protein A mass spectrometry for characterization of monoclonal antibodies.
abstract: Purification of antibodies is an important first step to produce material for in depth characterization of biotherapeutics. To reduce the resource burden incurred by protein purification, we developed a high throughput protein A affinity capture step coupled to inline mass spectrometry (PrA-MS). Our method enables both UV quantitation of antibodies and product characterization of an intact molecule with microgram quantities of material. When purification and analysis are coupled along with the low material demand, PrA-MS is widely applicable to protein characterization and is uniquely advantageous for moieties that rely on molecular stoichiometry. Two model systems were studied using PrA-MS and are presented here: (a) bispecific antibodies (bsAb) and (b) glycan engineered antibodies. In the bsAb samples, hetero- and homodimer species, along with partial molecule, were readily identified and quantified directly from harvested cell culture fluid (HCCF). In the glycan engineered antibodies, fully afucosylated, as well as asymmetrically and symmetrically fucosylated, glycans were identified from HCCF in experiments that utilized a small molecule inhibitor of fucosyltrasferase. The PrA-MS method represents a high throughput alternative to offline purification and product characterization that may be leveraged across the product lifecycle of engineered antibodies to enable rapid development and production of important therapeutics.
authors: Department of Analytical Sciences, Amgen Inc. , 1201 Amgen Court West, Seattle, Washington 98119, United States.
authors: Kenneth M
authors: KM
authors: Prentice
authors: Alison
authors: A
authors: Wallace
authors: Catherine M
authors: CM
authors: Eakin
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000032:analysis*; D000911:Antibodies, Monoclonal / Q000032:analysis*; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000295:instrumentation*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation; D003412:Cricetulus; D004867:Equipment Design; D006031:Glycosylation; D013058:Mass Spectrometry / Q000295:instrumentation*; D011134:Polysaccharides / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry*
chemical_list: D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D011134:Polysaccharides; D013205:Staphylococcal Protein A