Alternatives to Protein A Capture Chromatography

The first step in downstream bioprocessing is harvest via centrifugation or depth filtration to remove cell debris from the culture while minimizing product losses. The second step, capture, specifically removes the desired product from all other soluble components, e.g., spent culture media, byproducts, and both cell- and ingredient-derived impurities. The dominance of protein A resins for antibody capture is almost a given, but the emergence of non-immunoglobulin therapeutic proteins is causing a gradual shift toward other modalities.

“For monoclonal antibodies, capture is most easily achieved using protein A or protein G affinity media, which have high specificity for the Fc region of the mAb,” says Andrew Coffey, Ph.D., Applications Chemist at Agilent Technologies. “For other molecule types, finding an affinity medium with similar high specificity for the target protein can be more difficult. In some cases, it is possible to engineer a protein to incorporate a suitable label, such as poly His-tag or glutathione S-transferase.”

These species bind to affinity resins with specific functionalities such as an immobilized metal like Ni²⁺ in the case of poly His-tagging, or immobilized glutathione for GST-labeled proteins.

Development time, costs

Devising a novel purification process based on non-standard resins involves development time and costs, and some approaches might raise red flags with regulators for whom “the product is the process.” Scaleup development will surely involve the expenditure of even more resources, but innovative companies will continue trying because, as Coffey notes, “affinity media is usually the most expensive cost involved in downstream processing.”

When a suitable affinity medium is lacking, bioprocessors may turn to cation exchange chromatography for peptides and proteins, or anion exchange chromatography for oligonucleotides. “Purification based on ion exchange is not as specific and is usually better reserved for polishing steps,” Coffey says. Still other chromatographic options include size exclusion, hydrophobic interaction chromatography, and mixed mode chromatography. These techniques are often used for polishing—to remove trace contaminants and impurities, so bioprocessors are already familiar with them. “Membranes and fibers are also suitable for some purification steps where high resolution is not required,” he adds.

Platform purifications

Affinity chromatography will always be an option for therapeutic protein manufacturing, particularly for monoclonals, due to the possibility of designing more widely applicable platform purification processes, and its ability to reduce the number of downstream steps while improving product recovery.

Affinity resins are commercially available for non-antibody-based therapeutics, but according to Andrew Masters, Head of Business Development and Strategy at Purolite’s Bioprocessing Division, these are specific to groups of proteins, for example glycoproteins or a specific class of enzymes, rather than to a single molecule type. “Ligands used for group-specific affinity include heparin, calmodulin, lectins, and mimetics, which have been used to purify ATPases, adenylate cyclases, kinases, serine proteases, neurotransmitters, and DNA binding proteins to name a few. Compared with protein A, these resins typically have lower binding capacity for the target molecule and can be difficult to clean. Using them at manufacturing scale involves tradeoffs between performance, the potential for re-use, and overall process economics.”

In situations where affinity resins are not an option, developers apply standard purification protocols in which selectivities of various resins (e.g., ion exchange, HIC, mixed mode) are individually screened for their ability to capture the target.

“The goal of capture is to rapidly concentrate the target molecule, and remove bulk contaminants and proteases,” Masters adds, “which is why high capacity ion exchange resins, typically with incorporating sulfopropyl or quaternary amine chemistries, are commonly selected. Cation exchange is particularly useful for targets coming from microbial expression since the negatively charged ligand will not bind DNA or endotoxins.”

Protein A alternatives

As the diversity of antibody-based therapies increases, more affinity resins designed to bind to non-fc-containing fragments or domains are entering the market. “Improvements in dynamic binding capacity, milder elution conditions, and resistance to NaOH for clean-in-place operations are driving implementation of these resins into manufacturing processes,” Masters tells Biocompare. “Historically, some commercial mAb processes were developed using traditional ion exchange and hydrophobic interaction-based purification. However, the benefits of a platform approach—among them robustness and speed of development—and launch of highly productive, next-generation protein A resins, such as those with uniform particle size distributions, mean protein A will remain the gold standard for the foreseeable future.”

There may be roles for what Masters calls “disruptive purification technologies” such as membranes, monoliths, and recently nanofiber formats, especially for large-molecule purification where, due to the large target size, the total accessible binding surface area of the resin is limited to the outside of the bead. “These formats are currently being evaluated for monoclonal antibody manufacturing. However, they have limitations in capacity, applicable volumes, scale, and efficient fluid dynamics, resulting in a significant increase in consumption of process buffers.”

Affinity isn’t just for products

The design and validation of capture resins is costly and time consuming, but once the resins and processes are set, the potential to design platform purification trains more than compensates for the upfront effort. Capture strategies work for impurities as well, particularly when a particular expression system is known to generate specific, problematic host cell proteins.

In June 2022, German protein engineering firm Navigo Proteins introduced an affinity resin for capturing glycoprotein gp64 contaminants from the baculovirus insect cell expression system. Navigo has pioneered Precision Capturing^®, a technology that simplifies purification of non-Fc biologics by combining (and improving on) several chromatography steps into a single capture operation. The company’s Precision X ligand, for example, binds specifically to the gp64 contaminant. Since the target here is a contaminant, the resin is used in flow-through chromatography mode, where the contaminant binds to the resin but the recombinant product protein elutes without interacting with the resin. By focusing on a common expression system-based contaminant, Precision X can be used for more than one vaccine product. Novavax, for example, uses the resin to produce an influenza vaccine candidate.