Optimizing mAb Purification

Monoclonal antibody (mAb) purification typically includes sequential filtration, protein A affinity capture, and contaminant removal or polishing steps as needed. Under this general model, developers tweak purification unit operations to accommodate the peculiarities of the molecule. It’s now possible, however, through protein engineering, to design mAbs with sequence and structural features that improve general manufacturability and purification efficiency.

According to Desmond Schofield, EngD, Chief Business Officer at Swiss CRO evitria, "When we focus on antibodies, human IgG1 molecules for example, the primary purification-related liabilities in their sequences often involve additional cysteine residues and glycosylation sites."

Schofield explains that excess cysteines beyond those required to maintain critical disulfide bridges can disrupt the antibody’s natural structure, resulting in product instability or aggregation. “These extra cysteines often originate during animal-based antibody discovery campaigns,” he notes. “For example, rabbit-derived antibodies naturally have an additional disulfide bond in their kappa light chain, which, when reformatted into murine or human backbones, may inadvertently retain additional cysteines.”

Similarly, extra glycosylation sites, often introduced in complementarity-determining regions during antibody discovery, further complicate purification. "While these additional glycosylations might not necessarily impair antibody binding or function, they do introduce significant heterogeneity,” Schofield says, “which subsequently complicates downstream characterization and quality control."

Complexity: the fly in the ointment

Although traditional purification approaches remain effective for standard mAbs and biosimilars, they are less suitable for more structurally complex, emerging antibody variants such as bispecific antibodies, antibody fragments, and other novel mAb-inspired formats.

“Protein A remains dominant for antibody capture,” Schofield emphasizes, “but there's an exponentially growing need for innovative and complementary polishing techniques to achieve the selectivity required by complex antibody formats.”

This need arises primarily due to challenges in isolating the correct antibody species from chemically diverse production mixtures. “Purification must often distinguish desired heterodimers from a mixture that includes homodimers and other incorrectly formed byproducts,” Schofield says. “While ion-exchange chromatography has traditionally been the method of choice for resolving these species, novel affinity-based methods are becoming increasingly available. A notable example is tandem affinity chromatography using selective columns for kappa and lambda light chains, particularly beneficial when both chains are present in the same antibody.”

These advances in purification directly influence analytical and characterization strategies, creating new demands for validation. Schofield adds, "Standard antibodies historically benefit from straightforward and well-established analytical methods. However, as purification methods become more sophisticated for complex antibody formats, we observe a parallel innovation emerging in analytics to confidently characterize and confirm these novel molecules."

One class of mAb-related therapeutic, multispecific antibodies (msAbs), is gaining interest for its ability to modulate multiple biological targets. But what makes msAbs therapeutically interesting also presents unique purification issues due to their unique chemistry and novel product- and process-related impurities.

Sanofi recently described an approach  for removing product-related impurities by altering the binding affinity of light chains to the company’s Kappa Select and Protein L resins. In one case, the introduction of amino acid mutations in the constant light chain domain maintained antigen binding affinity while eliminating undesirable binding.

“These purification-enabling mutations (PEMs) did not affect the thermal stability or purity of the proteins tested,” the authors wrote.

In addition to designing-in purification via PEMs, Sanofi reported on an entirely affinity-based purification scheme employing standard protein A resin followed by Kappa Select and Protein L resins which, according to the paper, remove light chain mispaired and other product-like impurities.

Long live the platform!

The cost and time constraints inherent in mAb purification were once a source of upstream-downstream capacity mismatches and high material losses, resulting in purification earning the reputation as the primary “bottleneck” in mAb manufacturing. That is no longer true, according to Laurens Sierkstra, Ph.D., Senior R&D Director for Thermo Fisher Scientific’s purification and pharma analytics business.

“That was true for a long time, but advanced platform processes and strategies have alleviated that bottleneck. For example, utilizing protein A capture with larger vessels and columns, as deployed by some CDMOs and large biotech firms, better aligns downstream purification with upstream capacity.”

Further opportunities exist, moreover, to better align the cost contribution of downstream processing steps relative to the full process, Sierkstra tells Biocompare. Thermo Fisher Scientific, for example, is promoting membrane technologies to improve productivity for both capture and final polishing steps.

“In addition, we are seeing an increase in more complex mAbs where Protein A capture is ineffective or suboptimal, and for which manufacturers are looking for alternative platform approaches,” Sierska says.

Over the last ten years, Thermo Fisher Scientific has assembled a portfolio of scalable, platform-capable purification products specifically for those novel molecules such as CaptureSelect affinity resins targeting specific regions of complex mAbs such as kappa, lambda, CH1, or Fc regions.

“Platform technologies are still being used and strictly followed, as any deviation will result in additional process development and process/product qualification, which cost time and money,” Sierska adds, but tweaks are possible.

Polishing chromatography, which usually comprises two columns, may in some cases be reduced to one column.

“Improvements to the standard platform separations are also under investigation, for example through multi-column chromatography, continuous or semi-continuous processes. Elegant new innovations are also emerging that combine unit operations through membrane technology,” Sierska explains.

Protein A capture—arguably the most robust and proven downstream step—has been one area of intense innovation, resulting in the price of this resin leveling or in some cases falling.

“These improvements, combined with multi-column chromatography, have reduced the overall cost of the capture step,” Sierska says. Plus, affinity resin capture has been adapted to more-complex therapeutics. “But significant advances in non-affinity technologies have not yet been implemented as considerable development work is required to demonstrate their ability to deliver the right purity and yield. Incorporating affinity technology into membranes, to exploit the increased productivity of membranes versus resins, is being researched. However, these solutions are still in early development and not yet suited for large-scale manufacturing.”