Why low-pH elution fails on sensitive antibody modalities

Protein A affinity chromatography has run on the same elution chemistry for forty years: bind at neutral pH, elute at pH 3.0–3.5. The platform was built around full-length IgG, which tolerates a brief acid hold without losing structure or activity. For monoclonal antibodies destined for first-generation biologics pipelines, that worked — and still works.

The pipelines have changed. Bispecific antibodies, antibody-drug conjugates, viral vectors and cell-derived products now make up a growing share of clinical development. These formats are not built like a robust IgG. The assumption that an acid hold is a free operation is the assumption that quietly fails in the downstream — usually as aggregation in the eluate, sometimes as outright loss of activity, occasionally as a comparability problem that surfaces only in late-stage development.

What low-pH elution actually does

Protein A binds the Fc region of an antibody through a small set of interfacial residues, most of them histidines. Histidine has a side-chain pKa near 6, which is the basis for the classical elution mechanism: dropping the buffer pH below ~4 protonates these residues, disrupts the electrostatic and hydrogen-bonding network at the interface, and releases the bound antibody. The structural logic is elegant, and on a stable Fc it is also reversible.

What the mechanism does not control is what happens to the rest of the molecule. At pH 3.5 the entire antibody is exposed to conditions far from its native environment. Surface charges flip, internal salt bridges weaken, hydrophobic patches that the native fold keeps buried become accessible. For a thermodynamically stable IgG1, the molecule re-folds when neutralised. For less stable formats, parts of the journey are not fully reversible.

Where it breaks

Bispecific antibodies

Bispecific formats — knob-into-hole IgGs, asymmetric heterodimers, common-light-chain constructs — depend on a precise pairing of two different heavy chains. The interfaces that drive correct pairing are engineered onto an otherwise antibody-like scaffold, and they tend to be less stable than the native Fc dimer. Under acid stress the asymmetric interface relaxes first; on re-neutralisation, the molecule frequently does not return to the correctly paired conformation. The downstream signature is the same in most processes: a monomer peak that loses area, an aggregate peak that grows, sometimes a mis-paired species that elutes between the two.

Antibody-drug conjugates

ADCs carry a small-molecule payload coupled to the antibody through a chemical linker. Many of the linkers in current use — particularly the cleavable hydrazone and some maleimide chemistries — are themselves pH-sensitive. An acid hold during capture or polishing exposes the linker to conditions it was designed to be cleaved under, and a measurable fraction of payload falls off. The drug-to-antibody ratio drifts; the released payload then has to be cleared from the process, often by an extra polishing step. None of this is catastrophic on its own, but each acid step adds DAR variability that compounds across the run.

AAV and lentiviral vectors

Viral vector capsids are not antibodies, and they are not built for an acid environment. AAV capsids in particular show conformational changes below pH 4 — VP1/VP2/VP3 protein arrangement loosens, surface antigenic structure shifts, and at the low end of the typical Protein A elution range the capsid integrity itself becomes uncertain. For an AAV polishing step that aims to separate full and empty capsids, an upstream acid hold that has already compromised the full population works against the entire premise of the separation.

Fragile fusion proteins and cell-derived products

The pattern extends beyond named modality classes. Fc-fusion proteins where the fused partner is a flexible peptide or a small folded domain often unfold at pH 3.5 even when the Fc portion does not. Cell-derived therapeutics with surface-bound antibody fragments lose viability under acid exposure. The common thread is that classical Protein A elution is a chemistry designed around one molecule — robust full-length IgG — being applied to molecules it was never validated on.

The downstream cost

When acid elution drives aggregation, the downstream pays in three ways. The aggregate-removal polishing step works harder — typically a cation-exchange or HIC pass that now has to clear a larger and more heterogeneous aggregate fraction, with lower recovery. Yield drops, often by several percent per step. And for programmes that reach late-stage development, the acid-induced micro-heterogeneity becomes a comparability question: does the process at scale produce the same product as the process at bench? That question is harder to answer than to ask.

None of this shows up in a development summary table that only lists Protein A capture yield. The cost is paid by every step that follows.

What classical workarounds offer — and miss

The standard toolbox for taming acid elution is well known. Arginine, magnesium chloride or other chaotropic additives in the elution buffer raise the elution pH by reducing the strength of the Protein A–Fc interaction at higher pH. Step-elution profiles minimise the time the product spends at the lowest pH point. Immediate neutralisation into a Tris- or HEPES-based hold buffer shortens the acid exposure to seconds rather than minutes. Each of these helps. None of them changes the underlying mechanism: the product still has to survive a chemistry-driven release event.

For a robust IgG, the optimised acid step is good enough. For a bispecific where the heterodimer interface starts to relax at pH 4.5, raising elution pH from 3.5 to 4.0 reduces — but does not eliminate — the aggregation. The structural problem is that affinity is being released by changing the chemistry of the buffer, and the molecule cannot tell which part of the chemistry the change is supposed to act on.

A structural alternative: control by light, not chemistry

PureLight® is Lumatix's approach to that structural problem. The affinity ligand is built around a photoswitchable core: a small azobiaryl group integrated into a 3-helix-bundle scaffold that carries the target-binding interface. The chemistry of the elution buffer no longer controls release. A light pulse does.

Red light (630 nm) keeps the ligand in its high-affinity conformation. The product binds at native pH and ionic strength — the same buffer the harvest arrives in. Blue light (480 nm) triggers a reversible cis/trans isomerisation in the photoswitch; the ligand changes conformation, the affinity collapses, and the product elutes in whatever buffer you choose. No acid hold, no high-salt step, no chaotrope. The release event is optical, not chemical.

On Solaris® Protein A, our first product on this platform, the mechanism has been demonstrated over 100 IgG binding and elution cycles. The matrix underneath is the same hydrophilic cellulose monolith that powers MonoCore™; the difference is the ligand layer above it. The Beta evaluation programme is open by application — we are open to working with biotech and CDMO partners whose processes have a sensitivity-critical step worth running on light.

When PureLight matters — and when it does not

PureLight is not positioned as a replacement for classical Protein A on every process. For a robust full-length IgG running at commercial scale under cost-of-goods pressure, the optimised acid step is well understood, well validated, and economically hard to beat. Switching elution mechanisms on a working process buys little.

The fit is on sensitivity-critical modalities — bispecifics, ADCs, viral vectors, fragile fusion proteins, cell-derived products — where the acid step is the unit operation that quietly limits yield and complicates comparability. On those processes, neutral-pH elution is not a convenience. It is the difference between a downstream that works at scale and one that fights itself at every polishing step.

Outlook

Protein A is the first ligand on the PureLight platform; it is not intended to be the last. The 3-helix-bundle scaffold is designed to host different target-binding interfaces while keeping the photoswitchable release mechanism intact. The longer-term direction is a discovery platform for light-controlled affinity ligands against targets where no good affinity tool currently exists — which is most of the post-IgG biologics pipeline.

For now, the practical question is narrower. If your process has a step where the acid hold is the part you would change if you could, that is the step PureLight is built for.

Why low-pH elution is the wrong tool for sensitive modalities