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1.
Article in English | MEDLINE | ID: mdl-27212187

ABSTRACT

Antibody drug conjugates (ADCs) are complex therapeutic agents combining the selectivity of monoclonal antibodies and highly efficacious small molecule drugs to successfully eliminate tumor cells while limiting the general toxicity and side effects of the therapeutic to protect patient safety. One unique attribute critical to the safety of ADCs is the residual content of unconjugated small molecule drug present from either incomplete conjugation or degradation of the ADC. Typically for quality control assays, quantifying the amount of the free drug is performed through precipitation of the protein species using an organic solvent and then assaying the amount of free drug left in the supernatant. During the validation of an assay of this type for a maleimide based linker drug, issues were experienced with low and variable recovery in the spiked samples of the drug substance and drug product. A two-dimensional heart-cutting method coupling Size Exclusion Chromatography (SEC) with Reverse Phase (RP) chromatography was utilized to explore possible mechanisms leading to the low recovery of the free linker drug. The results of the investigation indicated that the spiked linker drug reacts with residual reactive groups on the ADC; a conclusion which was confirmed by the observed increase of average Drug to Antibody Ratio (DAR) determined by Hydrophobic Interaction Chromatography (HIC). Finally, several approaches were evaluated to minimize the recovery loss. Capping the residual reactive groups on the ADC with maleimide containing reagents effectively mitigated the low recovery issue.


Subject(s)
Chromatography, Gel/methods , Chromatography, Reverse-Phase/methods , Immunoconjugates/chemistry , Hydrophobic and Hydrophilic Interactions , Maleimides/chemistry
2.
MAbs ; 5(1): 86-101, 2013.
Article in English | MEDLINE | ID: mdl-23247543

ABSTRACT

Hydroxyl radical footprinting is a covalent labeling strategy used to probe the conformational properties of proteins in solution. We describe the first application of this high resolution technique for characterizing the structure of a therapeutic monoclonal antibody (mAb) dimer. As monitored by size-exclusion chromatography (SEC), therapeutic mAbs typically contain small amounts of a dimer species relative to the primary monomeric form in its drug substance or drug product. To determine its structural orientation, a sample enriched in an IgG1 mAb dimer was oxidized by hydroxyl radicals generated by exposure of the aqueous solution to synchrotron X-rays in millisecond timescales. The antibody monomer that served as a control was oxidized in a similar fashion. The oxidized samples were digested with trypsin and analyzed by RP-UHPLC-MS. The footprinting data show that peptides displaying decreased rates of oxidation (i.e., regions of increased protection) in the dimer are localized in the light and heavy chains of the Fab domain. The interface region for the monomers comprising the dimer was thus inferred to be between their Fab arms, allowing us to model two possible theoretical dimer orientations: a head-to-head, single arm-bound Fab-to-Fab dimer, and a head-to-head, double arm-bound Fab (') 2-to-Fab (') 2 dimer. Lower resolution fragment-SEC analysis of the dimer and monomer samples treated with papain or FabRICATOR enzyme provided complimentary evidence to support the Fab/Fab orientation of the IgG1 dimer.


Subject(s)
Antibodies, Monoclonal/chemistry , Hydroxyl Radical/chemistry , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin G/chemistry , Protein Footprinting/methods , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/therapeutic use , Chromatography, Gel , Dimerization , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin G/metabolism , Mass Spectrometry , Models, Molecular , Peptides/chemistry , Protein Conformation , Synchrotrons
3.
Biotechnol Bioeng ; 106(4): 627-37, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20229510

ABSTRACT

Pre-filtration using ion exchange membrane adsorbers can improve parvovirus filter throughput of monoclonal antibodies (mAbs). The membranes work by binding trace foulants, and although some antibody product also binds, yields > or =99% are easily achieved by overloading. Results show that foulant adsorption is dependent on pH and conductivity, but independent of scale and adsorber brand. The ability to use ion exchange membranes as pre-filters is significant because it provides a clean, well defined, chemically stable option for enhancing throughput. Additionally, ion exchange membranes facilitate characterization of parvovirus filter foulants. Examination of adsorber elution samples using sedimentation velocity analysis and SEC-MALS/QELS revealed the presence of high molecular weight species ranging from 8 to 13 nm in hydrodynamic radius, which are similar in size to parvoviruses and thus would be expected to plug the pores of a parvovirus filter. A study of two identical membranes in-series supports the hypothesis that the foulants are soluble, trace level aggregates in the feed. This study's significance lies in a previously undiscovered application of membrane chromatography, leading to a more cost effective and robust approach to parvovirus filtration for the production of monoclonal antibodies.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/isolation & purification , Filtration/methods , Parvovirus/isolation & purification , Culture Media/chemistry , Electric Conductivity , Hydrogen-Ion Concentration , Ion Exchange , Membranes
4.
Bioconjug Chem ; 19(8): 1673-83, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18637680

ABSTRACT

CD22 represents a promising target for antibody-drug conjugate therapy in the context of B cell malignancies since it rapidly internalizes, importing specifically bound antibodies with it. To determine the pharmacokinetic parameters of anti-CD22-MCC-DM1 and MC-MMAF conjugates, various approaches to quantifying total and conjugated antibody were investigated. Although the total antibody assay formats gave similar results for both conjugates, the mouse pharmacokinetic profile for the anti-CD22-MCC-DM1 and MC-MMAF appeared significantly different depending on the conjugated antibody assay format. Since these differences significantly impacted the PK parameters determination, we investigated the effect of the drug/antibody ratio on the total and conjugated antibody quantification using multiple assay formats. Our investigations revealed the limitations of some assay formats to quantify anti-CD22-MCC-DM1 and MC-MMAF with different drug load and in the context of a heterogeneous ADC population highlight the need to carefully plan the assay strategy for the total and conjugated antibody quantification in order to accurately determine the ADC PK parameters.


Subject(s)
Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacokinetics , Immunoconjugates/metabolism , Immunoconjugates/pharmacokinetics , Maleimides/metabolism , Maytansine/analogs & derivatives , Maytansine/metabolism , Oligopeptides/metabolism , Sialic Acid Binding Ig-like Lectin 2/immunology , Animals , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/immunology , Humans , Immunoconjugates/analysis , Immunoconjugates/immunology , Mice , Sensitivity and Specificity
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