Isotonic concentrations of excipients control the dimerization rate of a therapeutic immunoglobulin G1 antibody during refrigerated storage based on their rank order of native-state interaction.

Inert co-solutes, or excipients, are often included in protein biologic formulations to adjust the tonicity of liquid dosage forms intended for subcutaneous delivery. Despite the low concentration of their use, many of these excipients alter protein-protein interactions such as dimerization and aggregation rates of high concentration monoclonal antibody (mAb) therapeutics to varying extents during long-term refrigerated clinical storage, challenging the formulation scientist to make informed excipient selections at the earliest stages of development when protein supply and time are often limited. The objectives of this study were to better understand how isotonic concentrations of excipients influence the dimerization rates of a model mAb stored at refrigerated and room temperatures and explore protein sparing biophysical methods capable of predicting this dependence. Despite their prevalence of use in the biopharmaceutical industry, methods for assessing conformational stability such differential scanning calorimetry and isothermal equilibrium unfolding showed little predictive power and we highlight some of the assumptions and technical challenges of their use with mAbs. Conversely, measures of colloidal stability of the native-state such as preferential interaction coefficients measured by vapor pressure osmometry and solubility assessed by polyethylene-glycol induced precipitation correlated reasonably well with the mAb dimerization data and are most consistent with the excipients tested minimizing dimerization by interacting favorably with the residues comprising the protein-protein association interface.

A general approach to antibody thermostabilization.

Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). This approach allows both stabilization and maturation to affinities beyond those of the original antibody, as shown by the stabilization of an anti-HA33 antibody by approximately 10°C and affinity maturation of approximately 300-fold over the original antibody. Specificities of 10 antibodies of diverse origin were successfully transferred to the stable framework through CDR-grafting, with 8 of these successfully stabilized, including the therapeutic antibodies adalimumab, stabilized by 9.9°C, denosumab, stabilized by 7°C, cetuximab stabilized by 6.9°C and to a lesser extent trastuzumab stabilized by 0.8°C. This data suggests that this approach may be broadly useful for improving the biophysical characteristics of antibodies across a number of applications.

High affinity humanized antibodies without making hybridomas; immunization paired with mammalian cell display and in vitro somatic hypermutation.

A method has been developed for the rapid generation of high-affinity humanized antibodies from immunized animals without the need to make conventional hybridomas. Rearranged IgH D(J) regions were amplified from the spleen and lymph tissue of mice immunized with the human complement protein C5, fused with a limited repertoire of human germline heavy chain V-genes to form intact humanized heavy chains, and paired with a human light chain library. Completed heavy and light chains were assembled for mammalian cell surface display and transfected into HEK 293 cells co-expressing activation-induced cytidine deaminase (AID). Numerous clones were isolated by fluorescence-activated cell sorting, and affinity maturation, initiated by AID, resulted in the rapid evolution of high affinity, functional antibodies. This approach enables the efficient sampling of an immune repertoire and the direct selection and maturation of high-affinity, humanized IgGs.