ABSTRACT
The biological complexity associated with biotherapeutics such as antibody-drug conjugates (ADCs) requires extensive characterization to ensure product quality consistency, safety, and efficacy. ADCs generated via partial reduction of antibody interchain disulfide bonds result in a distribution of variants containing 0-8 conjugated drugs. Hydrophobic interaction chromatography (HIC) is a key analytical technique used to separate drug load variants of thiol linked ADCs and to calculate the average drug-to-antibody molar ratio (DAR). Salts present in HIC separations are not amenable to mass spectrometry (MS) detection, therefore confirmation of HIC peak identities have historically required offline fraction collection and subsequent MS analysis. We present a workflow comprised of three MS compatible two-dimensional liquid chromatography methods for higher throughput characterization of HIC peaks without manual fractionation. These methods are complementary and together provide DAR confirmation, identification of drug-load isomers, and localization of post-translational modifications to specific subunits. The results demonstrate an efficient mechanism for characterization of ADC HIC peaks and provided unique insight into differential HIC retention times based on conjugation sites and glycan structure.
Subject(s)
Chromatography, Liquid/methods , Immunoconjugates/chemistry , Immunoglobulin G/chemistry , Pharmaceutical Preparations/chemistry , Sulfhydryl Compounds/chemistry , Animals , CHO Cells , Cricetinae , Cricetulus , Immunoconjugates/analysis , Immunoconjugates/metabolism , Immunoglobulin G/analysis , Immunoglobulin G/metabolism , Pharmaceutical Preparations/analysis , Pharmaceutical Preparations/metabolism , Polysaccharides/chemistry , Sulfhydryl Compounds/analysis , Sulfhydryl Compounds/metabolismABSTRACT
Retention measurements involving 16 test solutes have been carried out for 38 type-A alkyl-silica columns and three bonded-zirconia columns. These measurements have been analyzed in terms of a model previously developed for type-B columns, so as to yield values of five column selectivity parameters (H, S*, A, B, C) for each type-A column. Overall differences in selectivity between type-A and -B columns can be related to the average values of H, S*, etc. for each column type. Compared to type-B columns, type-A columns provide generally stronger retention for carboxylic acids, while solutes that are more hydrophobic or less bulky are more retained on type-B columns. Hydrogen-bond acceptors (e.g. aliphatic amides) and cations (e.g. protonated bases) are strongly retained on type-A versus type-B columns. Compared to type-B columns, bonded-zirconia columns show much increased retention of cations and reduced retention of hydrogen-bond acceptors. Because of relatively large differences in the selectivity of bonded-zirconia, type-A, and type-B columns, it will prove difficult to find columns of different type (e.g. a type-A and a type-B column) which have equivalent selectivity. Type-A columns also tend to be more different from each other (in terms of selectivity) than is the case for type-B columns. As a result, the replacement of a given type-A column by an "equivalent" type-A column also appears unlikely, except for samples that do not contain ionized compounds.
Subject(s)
Chromatography, Liquid/instrumentation , Chromatography, Liquid/methods , Metals/chemistry , Silicon Dioxide/chemistry , Ethylamines/chemistry , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Kinetics , Molecular Structure , Sensitivity and Specificity , Zirconium/chemistryABSTRACT
Columns for reversed-phase HPLC (RP-LC) can be characterized by five, retention-related parameters: H (hydrophobicity), S (steric selectivity), A (hydrogen-bond acidity), B (hydrogen-bond basicity), and C (cation-exchange behavior). In the present study, values of the latter parameters have been measured for 92 type-B (low metals content)alkyl-silica columns and compared to column properties such as ligand length,ligand concentration, pore diameter, and the presence or absence of end-capping. With the exception of five columns of unusual design, retention factors, k, for 16 representative test compounds were correlated with values of H, S, etc., within an average +/- 1.2% (1 standard deviation, SD), suggesting that all significant solute-column interactions are recognized by these five column parameters. A single-valued function F(s) is proposed to measure differences in selectivity for any two RP-LC columns whose values of H, S, etc., are known. This allows the easy selection of columns whose selectivity is desired to be either similar to or different from a starting column, for application in either routine analysis or method development.
