A molecular-dynamics (MD) simulation was performed on these all-atom structures of the Fab domain with an explicit TIP3P water model.74 Each Fab domain was solvated in a cubic box with periodic boundary conditions in all 3 directions. Biobetters with increased stability against aggregation can therefore be generated in a rational manner, by either removing or masking the aggregation-prone region or crowding out protein-protein interactions. during mAb expression in position N195 is expected to mask the L154 residue (and its surrounding residues) and could potentially reduce the aggregation propensity of bevacizumab. A large set of residues were identified and further selective parameters were applied to ensure that feasible and efficient glycosylation would take place. Figure 3 summarizes the different criteria that were used (details in the Materials and Methods section) to select these glycosylation sites. After dismissing mutations leading to glycosylation site motifs that would not undergo glycosylation, as well as mutations potentially affecting protein structure, together with high SAP value residues, 8 residues were identified for site-directed mutagenesis to create potential glycosylation sites. Four different possible variants have been Potassium oxonate identified that will introduce a glycosylation site to mask the V110 high SAP value residue, whereas Potassium oxonate the 4 other mutations identified should permit the masking of 3 residues (L154, L180 and L201). With the aim of making our experiments more efficient and ensuring that we chose pertinent glycosylation sites, we did not introduce any glycosylation sites to mask the high SAP value residue V110. The V110 residue has been shown to be involved in aggregation, based on the observation that its mutation into lysine resulted in a 2.8-fold stabilization of bevacizumab Rabbit polyclonal to CyclinA1 against aggregation. Nevertheless, this reduction in aggregation is similar to that observed for variants L154D and L201K, and V110 has one of the lowest SAP scores among the above listed high SAP value residues, making its masking by a carbohydrate moiety less attractive. Open in a separate window Figure 3. Rational selection of residues for mutation to introduce glycosylation Potassium oxonate sites on the bevacizumab Fab domain. The five different questions can be asked in any particular order. Table 2. List of residues for glycosylation site engineering. Identification of variants which are likely to be glycosylated in the vicinity of high SAP regions. Residues in gray were not selected for the reasons described in the footnotes. immunogenicity assessment performed with the IEDB tools (Supplementary Potassium oxonate Information S4).62,63 In general, both approaches to disrupt potential aggregation-prone regions are effective strategies for improving mAb stability early on during drug discovery or for biobetter engineering.64,65 The removal and masking of APRs increases the stability of bevacizumab to the same level of the formulated drug. It is, therefore, likely that an appropriate formulation would further increase the stability of our biobetters. The masking and crowding of APRs by glycosylation motifs could potentially present further advantages over the removal of aggregation patches. The carbohydrate moiety might also stabilize the mAbs against other degradation routes such as hydrolysis, oxidation or deamidation. Even though experimental high throughput screening methods are being developed to identify developable mAbs,66,67 computational predictive approaches remain attractive and competitive due to their low cost and no requirement for materials.68 There is a clear incentive for the development of new platforms for high throughput screening of the developability and aggregation propensity of proteins with high accuracy, such as the sequence-based statistical model used in Lonza’s aggregation prediction tool.69 Their implementation early on during the discovery phase allows the reduction of costs, easier manufacturing, and the formulation of higher concentrations, opening the door for new delivery routes or reduced dose administration, all to the advantage of the patients and practitioners, with the benefits of a potentially safer drug and lower treatment costs. Materials and Methods Molecular simulation Several Fab MD simulations were performed, one of the wild-type of bevacizumab and one of each of the hyperglycosylated Fab variants (L118N, Q160N, Q160s and E195N). All three simulations were based on the crystal structure of bevacizumab, which was obtained from the RCSB PDB (PDB ID: 1BJ1).37 Hydrogen atoms were added to this structure at pH 7 using the PSFGEN plugin of the VMD.70 Topology and structure files for the hyperglycosylated Fab variants were generated using the Glycam website,71 assuming a G0 glycosylation pattern (GLYCAM notation: DGlcpNAcb1-2DManpa1-6[DGlcpNAcb1-2DManpa1-3]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1). For all simulations, the AMBER12SB72 and Glycam0673 force fields were used.