Molecular Design and Engineering

For biotechnology products, a key element in implementing QbD is engineering the molecule itself. A number of strategies are currently used by investigators to alter the properties of the molecule to achieve the desired balance among efficacy, stability, safety (such as immunogenicity), and manufacturability. These strategies include engineering the primary sequence to incorporate chemical and post-translational modifications for improved stability; recombinant and chemical fusion approaches for improved half-life and potency; and affinity maturation by phage display or transgenic mouse (e.g., xenomouse, Humax, and velocity mouse) technologies. Understanding the structure and functional attributes of therapeutic proteins, including monoclonal antibodies (MAbs), is key to developing the design space, because that understanding facilitates the selection of desirable quality attributes during molecular design while ensuring that bioactivity of the protein therapeutic is maintained.

Technologies based on combinatorial libraries and transgenic mice can generate a diverse panel of antibodies and protein-based candidates selected for binding and specificity for a target. Further selection and screening may involve recombinant cloning from hybridomas, humanization, and fusions (e.g., Fc or human serum albumin, PEGylation) and the use of stringent parameters like in vitro and in vivo assessment of biological activity. Other key factors in the selection of an efficacious molecule include affinity and the rate of dissociation from the target. Protein engineering also can be applied to increase tissue penetration and distribution.

Engineering the protein sequence is also used to reduce the risk of an immune response. Murine and chimeric antibodies used as human therapeutics frequently produce an immune response that can result in a reduction in or loss of activity and an unfavorable pharmokinetic (PK) profile requiring frequent administration of a drug. Humanization, transgenic mice, and phage display approaches make it possible to select antibody sequences bearing high homology to human germline sequences with the goal of reducing the risk of immunogenicity. Nevertheless, the risk of immune response to a fully human antibody and other self proteins necessitates screening therapeutic protein candidates for immunogenicity using in silico methods, and in vitro assays measuring T-cell responses. Sequence engineering also can be used to eliminate T-cell epitopes in the product.

Besides rational design for efficacy and safety, science-based hypothesis-driven approaches are used for selection and early engineering to achieve balance in the optimal physical and chemical properties of proteins with respect to product stability during and after manufacturing. A common observation during the development and commercialization of protein products is the susceptibility of certain amino acids (e.g., asparagine and methionine) to chemical modifications. Deamidation of asparagines and oxidation of methionines in regions important for activity can affect efficacy in some products. Sequence engineering also may be applied to minimize risks of chemical instability.

In summary, protein design and engineering are powerful tools for enhancing efficacy, modulating immunogenicity, altering proteolytic stability, introducing chemical modification sites, and improving expression. Rational design approaches make it possible to identify and engineer better protein therapeutic candidates right from the start, which combined with process optimization can pave the way for less frequent dosing, increased efficacy, decreased manufacturing costs, and an improved safety profile.^5–10