Repurposing Staphylococci for Biomanufacturing
Coagulase-positive staphylococci (CoPS) such as S. aureus possess mechanisms for evading the host immune system. For example, they can express Protein A (SpA), either anchored to their cell walls or secreted into the local environment, that binds and nullifies Igs to avoid detection and killing by host immune cells.2 Scientists have harnessed this binding affinity for other purposes, using SpA to purify human antibodies for therapeutic manufacturing. However, while SpA strongly binds three of four human IgG subclasses, it only strongly binds one of four canine IgG subclasses, with little to no binding of the other three. This makes it inefficient for purifying canine therapeutic antibodies.1
Seeking an alternative, Kazuhiko Ohashi and his team from Hokkaido University took a cue from the natural CoPS profile differences between dogs and humans. They recognized that while humans harbor S. aureus on their skin and mucosa, dogs host S. pseudintermedius instead. The SpA analog in S. pseudintermedius is SpsQ, and scientists previously revealed that this protein does bind canine IgGs.
Testing SpsQ
The question remained as to whether SpsQ binds canine IgGs with greater affinity than SpA. Ohashi and his colleagues first sequenced the spsQ gene expressed by S. pseudintermedius in order to produce recombinant SpsQ protein. They then designed an enzyme-linked immunosorbent assay (ELISA) to measure the concentration of the three canine IgG subclasses observed to bind SpsQ and SpA in cell supernatant.
The Hokkaido University team found no major difference in binding affinity between SpsQ and SpA for IgG-B (both high) and IgG-C (both zero). However, they noticed that SpsQ’s binding affinity for IgG-A and -D was significantly higher than SpA’s. Based on these results, the scientists hypothesized that they could use SpsQ as an affinity chromatography ligand to improve canine IgG purification efficiency.
Accordingly, they prepared resin columns immobilized with either SpsQ or SpA, and treated them with cell culture supernatants containing canine IgG-A, IgG-B, or IgG-D. They then analyzed recovered IgG purity using SDS-PAGE, finding that both SpsQ and SpA-immobilized resin removed impurities from IgG-containing cell culture supernatant samples.
Finally, they developed a custom ELISA to examine recovered IgG quantity. In this assay, the scientists coated microplates with antibodies from Bethyl Laboratories, a Fortis Life Sciences® company, to capture IgG-A and IgG-D (goat anti-dog IgG1), and IgG-B (sheep anti-dog IgG2). They then treated these microplates with biotinylated recombinant SpsQ and SpA. Next, Ohashi and his colleagues introduced horseradish peroxidase (HRP)-conjugated versions of anti-canine IgG1 or IgG2 antibodies to create an IgG-protein-IgG-HRP complex. Finally, they introduced TMB One Component Substrate from Bethyl Laboratories, which reacted with HRP to create an insoluble dark blue product that could be quantified via absorbance measurements.
Ohashi and his team recovered 1.5 to 1.7-fold more IgG-A and IgG-D from SpsQ-immobilized columns compared to SpA-immobilized columns. They saw no change in recovery when looking at IgG-B, an expected result since IgG-B showed similar binding affinity to both SpsQ and SpA.
Accelerating Antibody Development
Today, therapeutic antibodies are an essential part of human medicine. They are also a growing part of veterinary medicine, especially for companion animals such as dogs. This has created a demand for canine antibody mass production. However, canine antibodies are not perfect analogs to human ones, and human antibody production workflows do not translate verbatim for canine antibody production. Ohashi and his colleagues showed that SpsQ, developed from a dog commensal CoPS, binds canine IgGs more strongly than SpA, which is commonly used in human antibody production. SpsQ can improve antibody purification workflow efficiency, potentially contributing to the establishment of a cost effective and practical purification process for canine therapeutic antibodies.
References
- Takeuchi H, et al. Characterization of SpsQ from Staphylococcus pseudintermedius as an affinity chromatography ligand for canine therapeutic antibodies. PLoS One. 2023;18(1):e0281171.
- Boero E, et al. Natural human immunity against staphylococcal protein A relies on effector functions triggered by IgG3. Front Immunol. 2022;13:834711.
