Protocol for high-throughput cloning, expression, purification, and evaluation of bispecific antibodies

Abstract

Bispecific antibodies are a powerful new class of therapeutics, but their development often requires enormous amounts of time and resources. Here, we describe a high-throughput protocol for cloning, expressing, purifying, and evaluating bispecific antibodies. This protocol enables the rapid screening of large panels of bispecific molecules to identify top candidates for further development. For complete details on the use and execution of this protocol, please refer to Estes et al. (2021).

Keywords: Antibody; Biotechnology and bioengineering; High Throughput Screening; Protein Biochemistry; Protein expression and purification.

Graphical abstract

Figure 1

Schematic illustration of a bispecific Hetero-IgG molecule and corresponding Golden Gate Reaction (A) Schematic illustration of a bispecific Hetero-IgG molecule. HC, heavy chain; LC, light chain; VH, variable heavy region; VL, variable light region; CL, constant light region; CH1, constant heavy region 1; CH2, constant heavy region 2; CH3, constant heavy region 3. (B) Schematic illustration of DNA fragment design and Golden Gate Reaction. BsmBI, a type IIS restriction enzyme that cuts DNA outside of its recognition site; ccdB, a toxic gene that targets E.coli DNA gyrase.

Figure 2

Correlation of titer and ProA yield A total of 192 molecules in Hetero-IgG format were high-through expressed in HEK 293-6E cells. Expression titers were measured with conditional medium at day 6 post-transfection. ProA purification yields were determined by A₂₈₀ at day 7 post-transfection.

Figure 3

Correct species and potential undesired species for Hetero-IgG molecules MP, main peak; pre-MP, pre main peak; post-MP, post-main peak; HC, heavy chain; LC, light chain. HC mispairs are often lack of a covalent linkage in the hinge region. Therefore, HC mispairs are often dissolved by SDS in nrMCE and migrate like ½ species (pre-MP), while appear as MP in SEC. ∗, Abundance of species is sequence- and engineering-dependent.

Figure 4

Representative examples of nrMCE and aSEC methods for bispecific antibodies (A and B) A hetero-IgG was prepared and run on nrMCE (A) and aSEC (B) as described in steps 12 and 13, respectively. (C) aSEC profile showing Gel Filtration Standards (Bio-Rad). Approximately 18 μg of Standard was injected into a Zenix-C 300 (300 Å, 3 micron, 4.6 × 300 mm) SEC column as described in step 13.