Steven P. Piccoli

An integrated multiplatform bioanalytical strategy for antibody–drug conjugates: a novel case study

BACKGROUND The bioanalytical strategy for antibody-drug conjugates (ADC) includes numerous measurements integrally designed to provide comprehensive characterization of PK, PD and immunogenicity. This manuscript describes the utilization of reagents specifically tailored to an ADC with a microtubule polymerization inhibitor payload and cathepsin B cleavable linker. METHODS The PK strategy includes the evaluation of physiological levels of total antibody, active ADC, total ADC, antibody-conjugated payload and unconjugated payload. These data are evaluated in the context of target and antidrug antibody levels to elucidate bioactive ADC. RESULTS & CONCLUSION Herein, we discuss how this strategy has been applied and present our preliminary observations. Continuously evolving to meet pipeline demands, the integrated bioanalytical data will provide critical insights into the exposure-response relationship.

Anti-PEG antibody bioanalysis: a clinical case study with PEG-IFN-λ-1a and PEG-IFN-α2a in naive patients

BACKGROUND Extensive use of polyethylene glycol (PEG) in consumer products necessitates the assessment of anti-PEG antibodies (APAb). METHODS In clinical trials comparing PEG-IFN-λ to PEG-IFN-α, conventional bridge and direct assays were assessed. RESULTS & CONCLUSION The bridge assay detected IgM and IgG APAb reactive with common PEG sizes and derivatives at sufficient sensitivity, 15-500 ng/ml. Of subjects evaluated, 6% of PEG-IFN-λ and 9% of PEG-IFN-α subjects had persistent APAb while 60% of PEG-IFN-λ and 33% of PEG-IFN-α subjects had persistent anti-interferon antibodies (AIAb). Pre-existing APAb and AIAb prevalence was comparable (approximately 10% of subjects). APAb were earlier onset, less frequent, less persistent and lower titer than AIAb. No associated hypersensitivity events were reported.

Conjugated critical reagent characterization for ligand-binding assays: using MALDI-TOF-MS as an orthogonal tool to assess assay performance

Large-molecule biotherapeutics are forming an increasingly large percentage of emerging pharmaceutical pipelines. These molecules present specific challenges to the bioanalysts charged with measuring in vivo concentrations of the biotherapeutic. The challenges are typically met using ligand-binding assays in support of pharmacokinetic, pharmacodynamic and immunogenicity assays. Ligand-binding assays employ complex biological molecules that specifically recognize the biotherapeutic for quantitation. Generally, a minimum of one of these critical reagents must be chemically modified to generate a signal that is measured in the assay. Once chemically modified it is necessary to characterize the reagent prior to use in an assay. The concentration, purity and molar incorporation ratio of chemical modification are key characteristics. This article presents mass spectral techniques for determining the molar incorporation ratio. Case studies are provided to demonstrate the time and cost savings that can be realized with timely and detailed characterization of critical reagents for ligand-binding assays.

Development and characterization of antibody reagents to assess anti-PEG IgG antibodies in clinical samples

BACKGROUND Polyethylene glycol (PEG) is a polymer that can be conjugated with therapeutic proteins. Monitoring anti-PEG antibodies in human subjects may be required as part of immunogenicity assessment. The lack of well-characterized anti-PEG reagents have limited our understanding of anti-PEG humoral response. RESULTS Antibodies reactive to PEG were engineered with a human IgG1 Fc. Surface plasmon resonance and plate-based methods demonstrated that their binding was dependent on molecular weight (MW) of PEG. Specificity experiments using chemical analogs identified their specificity. CONCLUSION Affinity, specificity and MW of PEG are critical characteristics that impact interactions of anti-PEG antibodies with PEG. These attributes especially MW of PEG and the assay formats may impact the ability to detect anti-PEG antibodies.

Targeting an acid labile aspartyl–prolyl amide bond as a viable alternative to trypsin digestion to generate a surrogate peptide for LC–MS/MS analysis

BACKGROUND FGF21-AdPKE is a fusion protein and functionally inactivated in vivo by cleavage around the C-terminus. It is important to quantify the intact active protein in serum. RESULTS & DISCUSSION Taking advantage of a uniquely acid-labile aspartyl-prolyl amide bond, we developed an acid hydrolysis procedure based on heating FGF21-AdPKE in dilute formic acid to generate a surrogate peptide encompassing the last 17 amino acids at the C-terminus. The monkey serum samples were extracted with an immunocapture procedure with an antibody specific for AdPKE. The calibration range was 200-50000 ng/ml. The assay accuracy and precision were between 92.8-99.8% and 3.9-14.5%, respectively. The method was applied to analyze incurred serum samples from a cynomolgus monkey toxicokinetic study involving administration of FGF21-AdPKE. CONCLUSION A method of combining immunocapture and acid hydrolysis to quantify a therapeutic protein in biological fluids was developed.

Real-time Imaging of Protein Therapeutics Using Liquid Cell EM

Lynn M. DiMemmo1, A. Cameron Varano2, Jonathan Haulenbeek3, Madeline J. Dukes4, Steven P. Piccoli3 and Deborah F. Kelly2. . 1. Drug Product Science and Technology, Bristol-Myers Squibb Company, USA. 2. Virginia Tech Carilion Research Institute, Virginia Tech, Roanoke, VA 24016, USA. 3. Analytical and Bioanalytical Operations, Bristol-Myers Squibb Company, USA. 4. Application Science, Protochips, Inc., Raleigh, NC 27606, USA.