This is the first report to describe the stability engineering of a therapeutic antibody addressing non canonical cysteine residues and the design strategy reported here is generally applicable to other therapeutic antibodies and proteins. Keywords:antibody, aggregation, manifestation, stability, cysteinylation == Intro == Recombinant monoclonal antibodies (mAbs) represent an established and growing class of therapeutics, with more than 20 mAbs authorized for the treatment and prevention of disease. treatment and prevention of disease. It is PX-866 (Sonolisib) definitely becoming increasingly apparent, however, that not all candidate mAbs emerging from your drug discovery process are suitable for commercial development, when considering their expression levels, stability and product homogeneity. In particular, the phenomenon of protein aggregation is a common issue that compromises the manufacture, storage, administration, biological activity and security of biological drugs, including mAbs. In extreme cases, the consequences of biological drug aggregation can be severe. For example, aggregation of the anemia drug erythropoietin (EPO) was one of the factors implicated in EPO-derived immunogenicity that caused pure red cell aplasia, and subsequently fatalities, in patients.1Therapeutic antibody immunogenicity is usually rarely as severe as the case of EPO, 2but can still result in unfavorable outcomes, such as patients having to withdraw PX-866 (Sonolisib) from therapy.3The aggregation issue is somewhat exacerbated by the recent move, in the interests of patient convenience, toward subcutaneous self-administration of antibody drugs. In this case, the risk of aggregation is usually increased due to the high concentration of antibody required to fill a 1 mL syringe with an effective dose. Aggregation in the developing process can lead to unwanted heterogeneity in biological protein preparations. Pharmaceutical regulatory government bodies, such as the United States Food and Drug Administration (FDA), recommend that heterogeneity be closely monitored and characterized to ensure consistent drug activity between developing lots.4When antibodies are variable in their aggregation profile between production lots, costly monitoring and control procedures are necessary during the manufacturing process. There are many factors that can contribute to protein aggregation, including main PX-866 (Sonolisib) sequence, partial unfolding, post-translational modifications, hydrophobicity, charge, pH, heat, protein concentration and formulation. Because mAbs are large multidomain proteins, the factors that lead to aggregation are complex and are generally not well comprehended.5It is becoming standard practice in the industry to select lead antibodies based on both biological activity and aggregation profile. Aggregation propensity can be measured in a number of high throughput assays6-8and predicted via in silico tools.9,10If aggregation is recognized, formulation development is routinely used to minimize aggregation following a quality by design (QbD) approach. Nevertheless, there are limits to the level of improvement that can be achieved by formulation changes alone. Up to 50% of manufactured product is wasted in some cases.11Improved variable domain engineering strategies are important to address such issues early in the research phase of drug development to ensure the drug can meet the desired clinical performance. In the current study, we focused on an antibody targeting angiopoietin 2 (Ang2), a soluble ligand for the Tie2 receptor and an import regulator of pathological angiogenesis and inflammation. The correlation between Ang2 expression in tumors with regions of high angiogenic activity and poor prognosis in many tumor types makes Ang2 an ideal drug target. We previously generated a human anti-Ang2 antibody that neutralizes Ang2 binding to the Tie2 receptor in vitro and inhibits angiogenesis and tumor growth in vivo12and now is in clinical trials.13Antibody development was hampered, however, by poor expression and aggregation caused in part by a non-canonical, unpaired Cys residue in the antibody variable domain name. Surprisingly, this antibody emerged from a B cell hybridoma screening strategy that should in theory include an intrinsic selection for well-expressed, non-aggregating antibodies. Antibody stability engineering strategies reported in the literature have focused on improving our general understanding of the residues linked to stability9,14-17or using directed evolutionary strategies to identify aggregation resistant frameworks.18,19Here, we started with an antibody with significant expression and aggregation liabilities and used a rational design Cspg2 approach to engineer the variable domain name to reduce aggregation and improve expression. This is the first statement of stability engineering addressing non-canonical Cys residues in an antibody and the strategy reported here is applicable to other proteins with unpaired Cys residues to enhance stability and therapeutic use. == Results == == Characterization of Ang2 mAb == The variable region genes from an anti-Ang2 hybridoma were cloned into a full-length human IgG2 vector and expressed in mammalian cells. Initial data from your expression and purification highlighted that this yield was significantly lower than expected (Table 1), but the mAb was greater than 99% monomer by SEC-HPLC (data not shown). Sequence analysis of.
