Approaches to Resolve False Reporting in Neutralizing Antibody Assays Caused by Reagent Leaching from Affinity Capture Elution Solid Phase
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Insufficient drug tolerance presents a major challenge in the development of neutralizing antibody (NAb) assays for biotherapeutics. Sample pre-treatment using solid-phase extraction with acid dissociation (SPEAD) is widely reported to improve drug tolerance. In this paper, a case study is presented in which SPEAD was used in conjunction with a competitive ligand binding NAb assay format. A significant degree of biotin-drug conjugate leaching was observed resulting in the reporting of both false positive and false negative results in NAb assay. Mitigation steps have been evaluated to address drug/biotin-drug conjugate leaching. These steps included assessment of the streptavidin-coated plate in conjunction with biotin-drug conjugates at various biotin molar challenge ratios (MCR). In addition, an alternative method based on covalent capture of the drug on an aldehyde-activated plate was assessed. Both approaches were compared for the degree of drug/biotin-drug conjugate leaching during the second elution step of the SPEAD procedure. Moreover, the impact of various conditions on the assay performance was assessed, including elution pH, sample incubation time, and biotin MCR. For the covalent drug capture method, capture conditions were evaluated. Optimized conditions in both streptavidin capture and covalent capture methods enabled a significant reduction of drug/biotin-drug conjugate leaching. A streptavidin high binding capacity approach using biotin-drug conjugate with a MCR of 50:1 was chosen as the optimal method yielding a NAb assay with a fit for purpose sensitivity (153 ng/mL) and a drug tolerance of up to 50 μg/mL with 500 ng/mL PC.
KEY WORDSACE anti-drug antibody biotin-drug conjugate/drug leaching drug tolerance immunogenicity neutralizing antibody sample pre-treatment SPEAD
The authors thank Lee Walus, Renee Ramsey, and Glenn Miller for the preparation of critical reagents and Fengping Li for the LC-TOF analyses of the biotin-drug conjugates. The authors also thank Frederick McCush, Ying Wang, Alison Joyce, Liang Zhu, and Marcela Araya Roldan for helpful input.
This work was funded by Pfizer.
- 4.FDA. Guidance for industry. In: Immunogenicity assessment for therapeutic protein products; 2014.Google Scholar
- 9.Shankar G, Pendley C, Stein KE. A risk-based bioanalytical strategy for the assessment of antibody immune responses against biological drugs. Nat Biotechnol 2007;25(5):555–561.Google Scholar
- 10.Rosenberg AS. Immunogenicity of biological therapeutics: a hierarchy of concerns. Dev Biol. 2003;112:15–21.Google Scholar
- 13.Green D. Spontaneous inhibitors to coagulation factors. Clin Lab Haematol 2000;22 Suppl 1:21–25; discussion 30-2.Google Scholar
- 15.Hu J, Gupta S, Swanson SJ, Zhuang Y. A bioactive drug quantitation based approach for the detection of anti-drug neutralizing antibodies in human serum. J Immunol Methods 2009;345(1–2):70–79.Google Scholar
- 19.Xu W, Jiang H, Titsch C, Haulenbeek JR, Pillutla RC, Aubry AF, et al. Development and characterization of a pre-treatment procedure to eliminate human monoclonal antibody therapeutic drug and matrix interference in cell-based functional neutralizing antibody assays. J Immunol Methods. 2015;416:94–104.CrossRefGoogle Scholar
- 21.Gupta S, Devanarayan V, Finco D, Gunn GR 3rd, Kirshner S, Richards S, et al. Recommendations for the validation of cell-based assays used for the detection of neutralizing antibody immune responses elicited against biological therapeutics. J Pharm Biomed Anal. 2011;55(5):878–88.CrossRefGoogle Scholar
- 22.EMA. Guideline on Immunogenicity assessment of 6 biotechnology-derived therapeutic proteins. 2015.Google Scholar
- 24.Heitzmann H RF (1974) Use of the avidin-biotin complex for specific staining of biological membranes in electron microscopy. Proceedings of the National Academy of Sciences USA 71(9):3537–41.Google Scholar