Residues within 10 from the domain junction were energy minimized (Amber99). by agitation or thermal stress while still retaining bioactivity. Keywords:agitation, aggregation, antibody, computational, engineering, in silico, modeling, stability, thermal == Abbreviations == spatial aggregation propensity relative aggregation propensity monoclonal antibody immunoglobulin G variable fragment fragment antigen-binding complementary-determining regions == Introduction == The value of protein therapeutics in human medicine is well known. The large gains demonstrated in the marketplace are WAY-100635 a testament to the demand for antibody medicines, which is expected to increase.1However, manufacturing protein therapeutics is an expensive and complicated process involving the control of many parameters. Appropriate control of these MHS3 parameters is critical to producing a quality medicine. One compromise in quality that these biomolecules can experience is the phenomena of protein aggregation.2,3Aggregation is considered an undesirable phenomenon that leads to a decrease in available efficacious product, potential immunogenicity in the patient and a host of other potential issues.4,5Protein aggregation can occur at any point during or after manufacturing. Physical stresses on the protein post manufacturing such as elevated temperatures or agitation on shipping (transportation) can induce aggregation.6The extent that a protein can withstand a particular stress is related to its stability to that stress. An environment can be found to stabilize the protein, such as through formulation screening. However, it is also possible to engineer the protein to withstand a stress, conferring a specific type of stability.7 We have encountered an IgG2 (mAb_A) that exhibited good solubility up to 70 mg/mL, but when agitation stressed, experienced severe aggregation. In silico homology modeling indicated a large amount of exposed hydrophobicity with respect to hydrophilicity in the variable fragment (Fv). The absolute amount of exposed hydrophobic surface area was similar to many other in-house Fv domains, but the absolute amount of exposed hydrophilicity was lower than typical for in-house Fv domains. For instance, the hydrophobic surface area for all Fvs in this therapeutic project was within 4% of each other (with an average of 5665 square ngstroms). The hydrophilic surface area for all Fvs WAY-100635 was within 1.3% of each other except for the Fv of mAb_A. The hydrophilic surface area of the mAb_A Fv was 3226 square ngstroms whereas the average for the other Fvs was 4815 square ngstroms. Additionally, the exposed hydrophobicity was arranged such that large patches were formed on the surface of the mAb_A Fv. We hypothesized that the large hydrophobic patches in the Fv were responsible for agitation instability of the IgG2. The lack of surface hydrophilicity and presence of large hydrophobic patches needed to be addressed simultaneously. One strategy for disrupting the hydrophobic patches is by inserting charged or hydrophilic, or less hydrophobic, residues within the patches or by simply deleting the offending hydrophobic residues. This strategy is uncommon for this type of situation. Considering that the majority of the exposed hydrophobicity was in the complementarity-determining regions (CDRs), we suspected that the paratope structure would be too drastically modified using those methods and therefore changes such as these would be detrimental to antibody function. Therefore, instead of merely inserting or deleting residues, we chose the strategy of attempting to retain the majority of the tertiary structure by WAY-100635 substituting offending hydrophobic residues with either charged or hydrophilic or less hydrophobic residues. The spatial aggregation propensity (SAP) algorithm developed in Bernhardt Trout’s laboratory at Massachusetts Institute of Technology8identifies and quantitatively calculates the degree of exposed hydrophobic motifs. The SAP algorithm, as part of the Accelrys Discovery Studio Suite (San Diego, CA), identified many residues and motifs that could be relevant to conferring aggregation instability in mAb_A. We selected certain sites with high SAP scores for site-directed mutagenesis. The type of residue substitution performed at each position was guided by utilizing a somewhat homologous IgG2 (mAb_B) that bound to the same target as mAb_A. Antibody B was found to be much more agitation stable in a small-scale relative aggregation propensity (RAP) assay we developed in-house. The high throughput RAP assay allowed us to efficiently test the stability of many variants by monitoring antibody loss post agitation or thermal stress. Not only was mAb_B less aggregation prone than mAb_A, it also exhibited high bioactivity to the.