title: On-line coupling of size exclusion chromatography with mixed-mode liquid chromatography for comprehensive profiling of biopharmaceutical drug product.
abstract: A methodology based on on-line coupling of size exclusion chromatography (SEC) with mixed-mode liquid chromatography (LC) has been developed. The method allows for simultaneous measurement of a wide range of components in biopharmaceutical drug products. These components include the active pharmaceutical ingredient (protein) and various kinds of excipients such as cations, anions, nonionic hydrophobic surfactant and hydrophilic sugars. Dual short SEC columns are used to separate small molecule excipients from large protein molecules. The separated protein is quantified using a UV detector at 280 nm. The isolated excipients are switched, online, to the Trinity P1 mixed-mode column for separation, and detected by an evaporative light scattering detector (ELSD). Using a stationary phase with 1.7 μm particles in SEC allows for the use of volatile buffers for both SEC and mix-mode separation. This facilitates the detection of different excipients by ELSD and provides potential for online characterization of the protein with mass spectrometry (MS). The method has been applied to quantitate protein and excipients in different biopharmaceutical drug products including monoclonal antibodies (mAb), antibody drug conjugates (ADC) and vaccines.
authors.affiliation: Analytical R&D, Pfizer BioTherapeutics R&D Pharmaceutical Sciences, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA. yan.he2@pfizer.com
authors.forename: Yan
authors.initials: Y
authors.lastname: He
authors.forename: Olga V
authors.initials: OV
authors.lastname: Friese
authors.forename: Michele R
authors.initials: MR
authors.lastname: Schlittler
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.forename: Xun
authors.initials: X
authors.lastname: Yang
authors.forename: Laura A
authors.initials: LA
authors.lastname: Bass
authors.forename: Michael T
authors.initials: MT
authors.lastname: Jones
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D002850:Chromatography, Gel / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005079:Excipients / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D013058:Mass Spectrometry; D004364:Pharmaceutical Preparations / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet; D014612:Vaccines / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D000097:Acetonitriles; D000911:Antibodies, Monoclonal; D005079:Excipients; D004364:Pharmaceutical Preparations; D014612:Vaccines; C032159:acetonitrile