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Custom Development of ADA Antibodies

Immunogenicity of a drug refers to its ability, or that of its metabolites, to elicit an immune response or immune‑related events against the drug itself or related proteins. As research and development of new drugs in China—particularly therapeutic protein therapeutics—continue to expand, studies on immunogenicity are becoming increasingly extensive.

Background

According to the FDA’s latest guidance, the assessment of a drug’s immunogenicity is an integrated, risk‑based approach that typically spans the entire biologic drug development process, from preclinical stages through post‑marketing surveillance. Consequently, studies on drug immunogenicity must be conducted continuously throughout both preclinical and clinical development, serving as a critical component of these phases. Drug immunogenicity can elicit the production of anti‑drug antibodies (ADAs) and/or neutralizing antibodies (NAbs). Based on its established hybridoma platform, Xichang Bio is able to rapidly and efficiently generate custom positive control antibodies for ADA assays tailored to clients’ needs.

Service Content

	Process	Cycle	Delivery Standards
Immune and Serum Titer Assays • Pre-immunization blood collection • Balb/c mice in immunization groups 2–3 •Serum titer assay • Final blood collection from the animal	Blood collection before immunization	Day 1	Interim Report (including results of antiserum titer assays exceeding 1:30,000) Deliver the remaining antigen.
	First immunization (antigen plus Freund’s complete adjuvant)	Day 1
	Second immunization (antigen plus Freund’s incomplete adjuvant)	Day 14
	Third immunization (antigen plus Freund’s incomplete adjuvant)	Day 28
	Blood collection and ELISA assay	Day 35
	Fourth immunization (antigen plus Freund’s incomplete adjuvant)	Day 42
	Blood collection and ELISA assay	Day 49
	Immune challenge (antigen dissolved in PBS or saline)	3 days before integration
Cell Fusion and Screening •Cell fusion •Fusion Filtering • Cloning and Cell Expansion • Cell line cryopreservation	Cell Fusion and Screening	2-3 weeks	~~1–2 pairs of positive cell lines that can be paired (2 cryovials per cell line)~~ Interim Report (including screening data)
	Xenocloning and cell expansion	3-4 weeks
	Cell line cryopreservation	1 week
Specificity screening: • ELISA screening of cell clones that exhibit strong positivity for Fab or F(ab’)2 and either no reactivity or weak reactivity with the isotype control IgG1; • Competitive ELISA was used to screen for cell clones with neutralizing activity.
Antibody Production and Purification • Culture of two monoclonal hybridoma cell lines •Protein A/G–purified antibody •ELISA assay • Purity testing	Hybridoma cell culture	2 weeks	Two strains each produce 1–3 mg of monoclonal antibody, with a titer greater than 20,000. Final Experimental Report
	Antibody purification	1 week
	ELISA assay, SDS-PAGE analysis, and concentration determination	1–2 weeks
Note: 1. At each stage—antiserum screening, fusion and selection, subcloning and selection, and antibody purification—screening is performed using hIgG1 to eliminate cross-reactivity. 2. Antibody Delivery Standards: (1) 1–2 monoclonal hybridoma cell lines secreting anti-ADA antibodies ~~(Within a pair, one may be a neutralizing antibody and the other a non-neutralizing antibody.)~~ (2) Purity > 95% (SDS-PAGE); antibody concentration > 0.5 mg/mL; purified antibody titer by indirect ELISA > 20,000-fold dilution; cross-reactivity with the hIgG1 control antibody < 2%; (3) COA file.