Antibody purification
Most antibody purification is two steps: capture the molecule on an affinity resin, then polish it to remove aggregates and the remaining contaminants. The details differ by format, and several routine choices decide how much aggregate you end up reporting.
Capture
Protein A binds the Fc of human IgG1, IgG2 and IgG4 well and IgG3 poorly, and it is the default capture step for IgGs. It also binds some VH3-derived variable domains, which is why a subset of VHHs stick to Protein A without an Fc.
Protein L binds certain kappa light chain variable domains, making it useful for Fabs and scFvs whose light chain is compatible. Compatibility is sequence-dependent, so it is a resin to test rather than to assume.
Protein G binds a broader range of IgG subclasses and species, which is useful for non-human material.
IMAC captures His-tagged proteins and is the standard route for tagged VHHs and antigens. Watch for host proteins with histidine-rich surfaces, and expect to polish afterward.
The elution problem
Affinity capture on Protein A or Protein L elutes at low pH. Many antibodies are not happy there, and time at low pH is one of the main places aggregates are created during purification. Two habits help: neutralize as soon as the peak comes off, into a buffer chosen in advance rather than improvised, and keep the time between elution and neutralization short and consistent across a panel.
If a candidate is acid-sensitive, milder elution conditions exist, including higher-pH elution buffers and alternative resins. It is worth finding out early, because an acid-sensitive molecule will otherwise look like an aggregation-prone molecule in every downstream assay.
Polishing
Size exclusion chromatography separates monomer from high molecular weight species and is the usual polishing step at discovery scale. It also gives you a purity readout for free.
Ion exchange separates by charge and resolves charge variants and some clipped species that SEC misses.
Buffer exchange into the final formulation buffer is part of polishing, not an afterthought. The buffer a protein sits in changes its aggregation behavior, so a panel stored in mismatched buffers will produce a stability ranking that reflects the buffers.
What to report with the material
A purification is not finished until the material is described: concentration measured with the correct extinction coefficient, purity from a size-based method, integrity on reducing and non-reducing gels, and endotoxin if the protein is going onto cells. For a panel, report these per lot, because a difference between two candidates in a cell assay is often a difference in aggregate content or endotoxin rather than in biology.
Practical notes
- Filter before loading, and never load a sample that has been sitting cloudy.
- Keep the same resin, the same flow rate and the same elution and neutralization scheme across a panel.
- Freeze-thaw cycles count. Record them.
- Aggregates measured on day one can grow. If material sits before an assay, re-check it.
How we run it
We purify in-house with one scheme per panel, neutralize consistently, polish where the material needs it, and report concentration, purity and integrity for every lot.