Domain-based assays of individual antibody concentrations in an oligoclonal combination targeting a single protein

Abstract

Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development, including pharmacokinetic (PK), toxicology, stability, and biochemical characterization studies of such drugs. We have developed an antitoxin, XOMA 3AB, consisting of three recombinant mAbs that potently neutralize the known subtypes of type A botulinum neurotoxin (BoNT/A). The three mAbs bind nonoverlapping BoNT/A epitopes with high affinity. XOMA 3AB is being developed as a treatment for botulism resulting from BoNT/A. To develop antibody-specific assays, we cloned, expressed, and purified BoNT/A domains from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. mAb-specific domains were used to develop an enzyme-linked immunosorbent assay (ELISA) for characterization of the integrity and binding activity of the three mAbs in the drug product. An electrochemiluminescence bridging assay that is robust to interference from components in serum was also developed, and we demonstrate that it can be used for PK assays. This type of antigen engineering to generate mAb-specific domains is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that binds the same protein and is superior to anti-idiotype approaches.

Figure 1. Structure of BoNT/A and BoNT/A domains

BoNT/A consists of a heavy chain (HC, magenta) and a light chain (LC, yellow). The HC consists of the receptor binding domain (H_C) and the translocation domain (HN). The HC consists of a C-terminal domain (H_CC) and an N-terminal domain (H_CN).

Figure 2. Scalable domain purification methods

Three separate purification strategies were developed to purify the BoNT/A H_CC, H_CN, and LC-H_N domains at the required scale. See text for details.

Figure 3. Analysis of the purity of BoNT/A H_CN, Hcc and LC-H_N domains by SDS-PAGE

Coomassie-stained SDS-PAGE gels were run to characterize the purity of domain fractions. Molecular weight markers (MW) are in the left lane of each gel. Lanes 1–3 H_CC (Lane 1, H_CC after refolding from denatured inclusion bodies; Lane 2, excluded elution shoulder fractions showing prominent “ladder” containing improperly oxidized H_CC species identified by RP-HPLC (see Figure 5); Lane 3, H_CC main peak). Lanes 4–6 H_CN (Lane 4, clarified H_CN lysate; Lane 5, partially purified H_CN ion exchange pool; Lane 6, Ni-NTA IMAC pool). Lanes 7–8 LC-H_N (Lane 7, partially purified ion exchange pool; Lane 8, purified LC-H_N pool).

Figure 4. Analysis of purity and immunoreactivity of BoNT/A H_CN domain by SE-HPLC

A. The purity and presence of aggregation can be identified by the elution profile of BoNT/A H_CN. B. mAb B elutes before H_CN, due to its larger size. C–E. When H_CN and mAb B are mixed together, the H_CN peak disappears, indicating that it is near 100% immunoreactive. At higher molar ratios of H_CN, the mAb B peak disappears.

Figure 5. Analysis of the purity of BoNT/A H_CC by reverse phase HPLC (RP-HPLC)

Top panel: Two major H_CC peaks are recognizable. Bottom panel: Addition of reducing agent to H_CC results in disappearance of one of the peaks, indicating that the two peaks shown in the top panel represent disulfide-oxidized and reduced H_CC (see text for details).

Figure 6. Domain-specific ELISAs

Binding of mAb A (A), mAb B (B), and mAb C (C) to plates coated with either H_CC, H_CN, or LC-H_N. Binding of each mAb was only seen to plates coated with their respective domain. Binding of mAb A (D) mAb B (E) or mAb C (F) to plates coated with their respective domain compared to the binding of a combination of all three mAbs (XOMA 3AB).

Figure 7. Pharmacokinetics of mAbs A, B, and C in rats

The concentration-time curves of mAb A, mAb B and mAb C following a single intravenous administration of XOMA 3AB at 0.1, 1, 10 mg/kg. Each point represents the mean ± SD of serum drug levels from six (3 males and 3 females) or more animals. The data from male and female animals were combined.