Monoclonal antibody (mAb) fragmentation can be a widespread problem across the biotechnology industry and there is a current need to better understand the underlying principles. Here, we report an example of a high-purity human IgG1 mAb prepared from CHO cells exhibiting fragmentation that can be attributed to residual proteolytic enzyme activity. The concomitant occurrence of proteolytic and non-proteolytic peptide bond cleavage is shown and the respective fragmentation patterns characterized using high-resolution LC-MS. Fragmentation rates are monitored by SE-HPLC and SDS-PAGE over the pH range 4-6 and characterized in the presence and absence of pepstatin A, an inhibitor of acidic proteases. After 20 days at 408C, pH 4, $60% decrease in BIIB-mAb monomer peak occurred attributed to residual proteolytic activity. At pH 5, this value was $13%. These results have implications for formulation design studies and the interpretation of accelerated stability data. A simple method to screen for acidic protease activity using the proteolytic enzyme inhibitor pepstatin A is described.

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(2011) A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity, mAbs, 3:3, 273-288,

Bispecific antibodies (BsAbs) target multiple epitopes on the same molecular target or different targets. Although interest in BsAbs has persisted for decades, production of stable and active BsAbs has hindered their clinical evaluation. Here, we describe the production and characterization of tetravalent IgG-like BsAbs that combine the activities of allosteric and competitive inhibitors of the type-I insulin-like growth factor receptor (IGF-1R). The BsAbs, which were engineered for thermal stability, express well, demonstrate favorable biophysical properties, and recognize both epitopes on IGF-1R. Only one BsAb with a unique geometry, denoted BIIB4-5scFv, was capable of engaging all four of its binding arms simultaneously. All the BsAbs (especially BIIB4-5scFv) demonstrated enhanced ligand blocking over the single monoclonal antibodies (mAbs), particularly at high ligand concentrations. The pharmacokinetic profiles of two IgG-like BsAbs were tested in nude mice and shown to be comparable with that of the parental mAbs. The BsAbs, especially BIIB4-5scFv, demonstrated an improved ability to reduce the growth of multiple tumor cell lines and to inhibit ligand-induced IGF-1R signaling in tumor cells over the parental mAbs. BIIB4-5scFv also led to superior tumor growth inhibition over its parental mAbs in vivo. In summary, BsAbs that bridge multiple inhibitory mechanisms against a single target may generally represent a more effective strategy for intervention in oncology or other indications compared with traditional mAb therapy.

Background: Integrin ␣1 I domain undergoes conformational changes upon collagen binding. Results: Deuterium exchange was used to measure the effects of cations, collagen, or an antibody on the ␣1I solution structure. Conclusion: Full-length collagen and metal ions induce changes that differ in key aspects from previously proposed models for ␣1I activation. Significance: These studies support a new model for integrin I domain activation.