Titer and integrity QC
Every assay downstream assumes it knows what is in the tube. Titer and integrity QC is how that assumption gets earned. For a discovery panel it is a handful of measurements, run the same way on every lot, and it is the cheapest insurance in the whole campaign.
Concentration
Absorbance at 280 nanometers is the standard, and it is only as good as the extinction coefficient used. Compute the coefficient from the sequence rather than reusing a generic antibody value, especially for VHHs and engineered constructs, where tryptophan and tyrosine content varies.
Scattering from aggregates inflates absorbance readings. If a sample looks concentrated and behaves dilute, check it on a size-based method before believing the number.
Purity and size
SDS-PAGE, run reduced and non-reduced, is the fast check. Reduced tells you the chains are the right size. Non-reduced tells you whether they are assembled and whether there is clipping or covalent aggregation. For a panel, run them on one gel so the comparison is real.
CE-SDS is the quantitative version of the same information and is what a program will want once decisions depend on percentages.
SEC gives the monomer percentage and shows high molecular weight species. It is the number that matters most for anything that will be dosed or stored.
Identity
Intact mass by LC-MS confirms you made what you designed, catches unexpected modifications, and is the only routine method that will tell you a signal peptide was cleaved in the wrong place or that a construct picked up a mutation that the plasmid sequencing missed.
For an antibody panel, mass also confirms glycosylation status when that matters, and it confirms the drug to antibody ratio after conjugation.
Endotoxin
Anything going onto cells needs an endotoxin measurement. Endotoxin produces effects in cell assays that are easy to mistake for biology, and the level varies between lots from the same expression run.
Making the numbers comparable
- Measure every lot in a panel by the same methods on the same day where possible.
- Report values per lot, with the lot identifier carried into the assay data.
- Record storage buffer, concentration and freeze-thaw count with the result.
- Re-check material that has been stored before it goes into a decision-grade assay.
What this catches in practice
Panels routinely contain one or two candidates that express poorly, come out clipped, or carry a much higher aggregate fraction than the rest. Without QC, those candidates go into the assay and generate results that look like weak binding or like toxicity. With QC, they are either fixed or flagged, and the assay result means what it appears to mean.
How we run it
We report concentration, purity, integrity and, where the work needs it, mass and endotoxin for every lot, with the lot identifier carried into every downstream data file.