Potential aggregation prone regions in biotherapeutics: A survey of commercial monoclonal antibodies

Abstract

Aggregation of a biotherapeutic is of significant concern and judicious process and formulation development is required to minimize aggregate levels in the final product. Aggregation of a protein in solution is driven by intrinsic and extrinsic factors. In this work we have focused on aggregation as an intrinsic property of the molecule. We have studied the sequences and Fab structures of commercial and non-commercial antibody sequences for their vulnerability towards aggregation by using sequence based computational tools to identify potential aggregation-prone motifs or regions. The mAbs in our dataset contain 2 to 8 aggregation-prone motifs per heavy and light chain pair. Some of these motifs are located in variable domains, primarily in CDRs. Most aggregation-prone motifs are rich in beta branched aliphatic and aromatic residues. Hydroxyl-containing Ser/Thr residues are also found in several aggregation-prone motifs while charged residues are rare. The motifs found in light chain CDR3 are glutamine (Q)/asparagine (N) rich. These motifs are similar to the reported aggregation promoting regions found in prion and amyloidogenic proteins that are also rich in Q/N, aliphatic and aromatic residues. The implication is that one possible mechanism for aggregation of mAbs may be through formation of cross-beta structures and fibrils. Mapping on the available Fab-receptor/antigen complex structures reveals that these motifs in CDRs might also contribute significantly towards receptor/antigen binding. Our analysis identifies the opportunity and tools for simultaneous optimization of the therapeutic protein sequence for potency and specificity while reducing vulnerability towards aggregation.

Figure 1

(A) Sequence alignment of light chains of commercial mAbs. The letters at the end of the mAb brand name are used to indicate its source. ‘u’ for human, ‘zu’ for humanized, ‘xi’ for chimeric, ‘o’ for mouse and ‘ov’ for sheep mAb. For molecular name of each mAb, refer to Table 1. All cysteine amino acids are highlighted in green. The three CDRs are highlighted in yellow. The predicted aggregation-prone segments are in red letters. (B) Sequence alignment of light chains of the twenty randomly selected non-commercial mAbs. Highlighting has the same meaning as in (A).

Figure 2A

Sequence alignment of heavy chains of commercial mAbs. ‘G₁’ or ‘G₂’ after the mAb brand name are for IgG1 and IgG2, respectively. For molecular name of each mAb, refer to Table 1. Highlighting scheme is same as Figure 1. In addition, the generic hinge region is highlighted in blue. Cysteine amino acids in hinge are highlighted in red. Note the hinge region of Vectibix is shown with a canonical IgG2 hinge. The mAbs except Remicade are grouped according to isotype and ordered based on degree of humanization within each group.

Figure 2B

Sequence alignment of light chains of the twenty randomly selected non-commercial mAbs. Highlighting has the same meaning as in (A).

Figure 3

The TANGO and PAGE profiles for trastuzumab (Herceptin) mAb. (A) Light chain profile. X-axis shows sequence number. Left y-axis and blue curve are for PAGE Z score. Right y-axis and black curve are for TANGO aggregation percentage. The red horizontal line indicates Z score = 1.96. (B) Heavy chain profile. Axes and line styles have same meaning as in (A).

Figure 4

Cartoon representation of the superimposed Fab structures of bevacizumab (Avastin), alemtuzumab (Campath), cetuximab (Erbitux), trastuzumab (Herceptin), basiliximab (Simulect) and palivizumab (Synagis). All light chains are in red and heavy chains in blue.

Figure 5

Structure of trastuzumab (Herceptin) Fab in complex with extracellular domain of HER2 is shown in cartoon representation. Green chain is HER2. Light and heavy chains of trastuzumab (Herceptin) are shown in gray and blue, respectively. The predicted aggregation-prone motifs are highlighted with different colors. The magenta segment is LLIYSASFLY in CDR L2 and the red is YCQQHY in CDR L3. The two motifs SVFIFP and VVCLL in L_C are in cyan and green, respectively.