Remediating agitation-induced antibody aggregation by eradicating exposed hydrophobic motifs

Abstract

Therapeutic antibodies must encompass drug product suitable attributes to be commercially marketed. An undesirable antibody characteristic is the propensity to aggregate. Although there are computational algorithms that predict the propensity of a protein to aggregate from sequence information alone, few consider the relevance of the native structure. The Spatial Aggregation Propensity (SAP) algorithm developed by Chennamsetty et. al. incorporates structural and sequence information to identify motifs that contribute to protein aggregation. We have utilized the algorithm to design variants of a highly aggregation prone IgG2. All variants were tested in a variety of high-throughput, small-scale assays to assess the utility of the method described herein. Many variants exhibited improved aggregation stability whether induced by agitation or thermal stress while still retaining bioactivity.

Keywords: CDR, complementary-determining regions; Fab, fragment antigen-binding; Fv, variable fragment; IgG, immunoglobulin G; RAP, relative aggregation propensity; SAP, spatial aggregation propensity; aggregation; agitation; antibody; computational; engineering; in silico; mAb, monoclonal antibody; modeling; stability; thermal.

Figure 1.

Locations of the engineered sites in the Fv of mAb_A. The variable light domain is rendered in orange ribbon. The variable heavy domain is rendered in magenta ribbon. The CDRs are rendered in dark blue ribbon. Only the residues rendered in CPK were substituted as described in Table 2. CPK Residues of the same color belong to the same SAP motif. Each SAP motif is indicated by the molecular surface of the same color to which the CPK residues belong. The upper image was rotated 90° toward the viewer to accomplish the orientation shown on the lower image where the CDRs are oriented toward the viewer.

Figure 2.

The relationship between transient expression antibody titer and either agitation stability (A) or thermal stability (B). Monoclonal antibody ‘A’ (mAb_A) is indicated by the black circle and mAb_B is indicated by the black square. The variants of antibody ‘A’ are designated by the diamonds. The color of the diamonds represents the quantity of substitutions as compared to antibody ‘A’. Red, green, gray, brown, blue, orange, yellow and purple represents 1 through 8 substitutions in a variant, respectively. Antibody variants with improved thermal stability usually exhibited higher expression titer. The same trend was not as evident for agitation stability.

Figure 3.

A cation exchange (CEX) column elution profile of the parent antibodies (A) and an antibody mixture (B). The CEX profile on the parent antibodies shown in (A) shows the dramatic stability difference between mAb_A and mAb_B post agitation (vortexing). The unstressed sample chromatogram is indicated by the black trace. The red and blue traces are from the same sample stressed by agitation for either 10 or 30 minutes, respectively. The decrease in peak height reflects sample loss due to protein aggregation induced by the stress. Shown in CEX profile (B) are 2 variants of mAb_A (14 and 41) along with the mAb_B control and 3 other stability standards (STD 1–3). Color coding in (B) is the same as in (A). Shown in this example chromatogram is mAb_A_41 with improved agitation stability over mAb_A_14, but neither exhibited agitation stability to the extent of mAb_B.

Figure 4.

Agitation and thermal stability of all purified antibodies. Monoclonal antibody ‘A’ (mAb_A) is indicated by the black circle and mAb_B is indicated by the black square. The variants of antibody ‘A’ are designated by the diamonds. The color of the diamonds represents the quantity of substitutions as compared to antibody ‘A’. Red, green, gray, brown, blue, orange, yellow and purple represents 1 through 8 substitutions in a variant, respectively. Plot (A) shows that engineered mAb_A variants with a higher number of residue substitutions often conferred improved agitation stability. However, those variants with improved agitation stability did not always exhibit improved thermal stability as shown in plot (B). The variants in the lower-left section of the plot did exhibit improved stability to both stresses as compared to the mAb_A parent. The quantity of substitutions indicated by the ordinate only applies to the engineered variants of mAb_A.

Figure 5.

Antibodies that bound in the Biacore assay. The black circle represents mAb_A, the black square represents mAb_B and the mAb_A variants are represented by the diamonds. The color of the diamonds represents the number of residue substitutions as compared to the mAb_A parent (as described in previous figures). Marker size from smallest to largest indicates the binding affinity range from 0.18 nM to 12.40 nM. The dotted line is the upper limit of quantitation of the biological assay, 3.33 nM. The 3 labeled variants exhibited improved agitation stability and sub-100 pM biological activity. The only substitution they all had in common was I30T in framework 1 of the VH.

Figure 6.

Location and type of mAb_A substitutions in variant 42. The 3 substitutions (T39D in green CPK, I30T in yellow CPK and L9D in aqua CPK) were effectively distributed on the Fv to remediate much of the aggregation experienced by the mAb_A parent. Their motifs are indicated by the translucent molecular surfaces. These 3 substitutions also caused minimal impact to biological activity as compared to the parent. The 2 large SAP motifs indicated by the red and blue molecular surfaces at the top-central region of the Fv conferred minimal instability on agitation in this construct.