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Lexington, MA, United States

Wei C.,Pfizer | Zhang G.,Pfizer | Zhang G.,Research and Nonclinical Development | Clark T.,Pfizer | And 5 more authors.
Analytical Chemistry | Year: 2016

The reactive thiol of cysteine is often used for coupling maleimide-containing linker-payloads to antibodies resulting in the generation of antibody drug conjugates (ADCs). Currently, a numbers of ADCs in drug development are made by coupling a linker-payload to native or engineered cysteine residues on the antibody. An ADC conjugated via hinge-cysteines to an auristatin payload was used as a model in this study to understand the impact of the maleimide linkers on ADC stability. The payload was conjugated to trastuzumab by a protease-cleavable linker, maleimido-caproyl-valine-citruline-p-amino-benzyloxy carbonyl (mcVC-PABC). In plasma stability assays, when the ADC (Trastuzumab-mcVC-PABC-Auristatin-0101) was incubated with plasma over a 144-h time-course, a discrepancy was observed between the measured released free payload concentration and the measured loss of drug-to-antibody ratio (DAR), as measured by liquid chromatography-mass spectrometry (LC-MS). We found that an enzymatic release of payload from ADC-depleted human plasma at 144 h was able to account for almost 100% of the DAR loss. Intact protein mass analysis showed that at the 144 h time point, the mass of the major protein in ADC-depleted human plasma had an additional 1347 Da over the native albumin extracted from human plasma, exactly matching the mass of the linker-payload. In addition, protein gel electrophoresis showed that there was only one enriched protein in the 144 h ADC-depleted and antipayload immunoprecipitated plasma sample, as compared to the 0 h plasma immunoprecipitated sample, and the mass of this enriched protein was slightly heavier than the mass of serum albumin. Furthermore, the albumin adduct was also identified in 96 h and 168 h postdose in vivo cynomolgus monkey plasma. These results strongly suggest that the majority of the deconjugated mc-VC-PABC-auristatin ultimately is transferred to serum albumin, forming a long-lived albumin-linker-payload adduct. To our knowledge, this is the first report quantitatively characterizing the extent of linker-payload transfer to serum albumin and the first clear example of in vivo formation of an albumin-linker-payload adduct. © 2016 American Chemical Society. Source

Shen Y.,Research and Nonclinical Development | Zhang G.,Research and Nonclinical Development | Yang J.,Research and Nonclinical Development | Qiu Y.,Research and Nonclinical Development | And 3 more authors.
Analytical Chemistry | Year: 2015

The formation of antidrug antibodies (ADA) can interfere with the accurate quantitation of therapeutic proteins, leading to significantly underestimated drug concentrations and confounded pharmacokinetic (PK) data interpretation. Although highly desirable, development of ADA-tolerant bioanalytical methods enabling unbiased measurement of both free and ADA-bound drug presents a considerable challenge. We report herein the development and validation of a robust LC-MS assay capable of quantifying therapeutic protein immunoglobulin A1 protease (IgAP) in human serum in the presence of pre-existing anti-IgAP antibodies. The procedure included sodium dodecyl sulfate (SDS) denaturation and chemical reduction of serum proteins to dissociate ADA-drug bindings, followed by tryptic digestion of protein pellets and subsequent LC-MS analysis of the surrogate IgAP peptide using stable isotope labeled peptide internal standard. Substantial enhancements in the sensitivity and selectivity were achieved by the combination of online two-dimensional reversed-phase LC (2D-LC) operated in high and low pH buffers, respectively, for efficient enrichment and quantitation of the surrogate peptide by multiple-reaction monitoring (MRM) mass spectrometry. Unlike ligand-binding assay, our method is not prone to interferences from ADA, allowing accurate and precise measurement of the IgAP in the range of 0.05 to 10 μg/mL in 25 μL of human serum with a wide range of anti-IgAP antibody levels. The intra- and inter-run precision (coefficient of variation (CV%)) was within 11.5% and 10.5%, respectively, and the bias was within ±7.1% for all quality control (QC) concentrations. With little modification, the described method can readily be applicable to the quantitation of other biotherapeutic proteins in the ADA-positive clinical matrices. © 2015 American Chemical Society. Source

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