Figure 1: SDS-PAGE analysis of the basic/neutral IgMs purified on Nuvia S and CHT-II-40. M: Protein marker, L: Load, FT: Flowthrough, EP: Eluted protein, HC: Heavy chain, LC: Light chainp

Immunoglobulin M (IgM) antibodies are currently a hot target for in vitro diagnostics and therapy for various maladies, including cancer and infectious diseases. The IgM backbone is essentially formed by a crosslinkage of five or six IgGs. However, IgMs are structurally and biochemically much more complex than IgGs. For one, IgMs are more heavily glycosylated than IgGs. They are very sensitive to pH conditions, making them highly susceptible to degradation or precipitation. Therefore, they are soluble in a narrow range of conditions. This makes their downstream purification difficult.

IgM purification is made even more complex by their large size, labile nature and complex physicochemical properties. The diffusion constant for IgMs is about half of IgGs, which means IgMs need half the flow rate to attain similar capacity and separation performance on a resin. Hence, the purification techniques used for IgGs cannot be translated directly to IgMs. In addition, affinity chromatography, which is commonly used for IgG purification, also cannot be successfully applied to process-scale IgM purification. The acidic conditions required for elution of targets from an affinity resin are too harsh and adverse for IgM stability.

Therefore, a nonaffinity-based strategy can be explored for basic/neutral IgM purification, utilizing an ion exchange and a mixed-mode resin. Optimization of the buffer pH and conductivity lead to a protocol that could minimize both product- and process-related impurities and produce a pure, stable fraction of IgM.

Purification of basic/neutral IgM antibodies
Purification workflows typically involve multiple steps to ensure high purity of the target. This application incorporated capture and polish steps in the purification workflow. A capture resin must ideally have high dynamic binding capacity (DBC) and the capability to interact with the target molecules more strongly than the impurities to ensure a relatively pure and concentrated eluate for the next step in the workflow. Ion exchange (IEX) resins have been incorporated at different steps of IgG and IgM purification processes in the past. Typically, IgMs are more charged than IgGs, leading to higher retention on IEX resins. As such, we chose a strong cation exchange (CEX) resin—Nuvia S—for the capture IgM purification. The use of the Nuvia S Resin eliminated the need for potential purification of any leached Protein A from downstream samples, which is a limitation of using Protein A affinity resin for capture. Hydroxyapatite-based resins have been shown to be very valuable in IgM purification in the past, owing to its strong binding to IgMs at physiological pH relative to sample contaminants. CHT Ceramic Hydroxyapatite (Bio-Rad) was therefore selected for the polish purification. CHT is a mixed-mode media; biomolecules bind to CHT either via CEX or affinity interactions, or both.

Capture purification
Three basic/neutral IgMs were expressed in an HEK transient expression system with isoelectric points (pI) ranging from 7.0 to 7.5. The buffer pH at all points during the purification was kept lower than the pI, making the IgMs positively charged and capable of binding with the CEX resin. Capture was initially tested at pH 5.0 with a constant salt gradient of 500 mM NaCl. Although the impurities—transferrin (TF), serum albumin (SA), free IgM light chain (LC), and monomeric IgMs—were detected in the same samples as the multimeric IgMs, the fractions were cleaner for the IgM with a pI of 7.0. To test the hypothesis that a buffer pH closer to the pI of the IgMs would lead to better sample purification, a pH 1.5 units lower than the pI of the IgMs was used, with a constant linear gradient of 500 mM NaCl. The relatively pure IgMs were then purified in bind/elute mode using Nuvia S Resin. Capture and partial purification at a pH so close to the pIs prevented the exposure of IgMs to the harsh conditions required for Protein A affinity based elution, thereby not jeopardizing the stability of the IgM. Additionally, the use of a higher pH for the elution buffer—close to the target pI—ensured that a low conductivity buffer could be used. This eliminated the need for downstream dilution before loading onto the next column.

Polish purification
CHT Ceramic Hydroxyapatite, Type II, 40 µm (CHT-II-40) Mixed-Mode Media was used for further purification of the basic/neutral IgM antibodies. The calcium groups on CHT can bind to the carboxyl groups on biomolecules via a CEX interaction, and the phosphate groups on CHT can bind to the amino groups on biomolecules via affinity interactions. Both types of interactions can also occur simultaneously. CHT thereby offers multiple modes by which IgMs can be bound and retained on the column, while impurities can be separated out in the flowthrough. A salt gradient is typically used for breaking the CEX interaction, whereas a phosphate gradient can break the affinity interaction. Since hydroxyapatite binds IgM strongly at physiological pH, conductivity, mild, non-denaturing conditions can be used for elution. Optimization studies showed that both salt and phosphate gradients were required for differential purification of IgMs from the remnant impurities. We achieved complete purification of the three basic/neutral IgMs with a 1 M NaCl and 500 mM NaPO4 gradient (Figure 1). The optimized protocol yielded purified fractions of all three IgMs tested, with minimal product- and process-related impurities.

In conclusion, a nonaffinity-based purification process utilizing a CEX resin and a mixed-mode media was developed for successful purification of basic/neutral IgMs. The workflow involves only two steps and requires a narrow range of optimized conditions, making it good for scalable process purification. Using mixed-mode media with high capacity and selectivity also helped overcome the affinity-based challenges of low binding and/or recovery during IgM purification. This strategy is simple, scalable and efficient and, thus, well suited for purification of diagnostic IgMs. Another polish step can be added to the process to generate IgMs with higher purity for therapeutic purposes. Such purification platforms can also be extended to purifying a diverse group of targets, including acidic IgMs, Fab fragments, diabodies and bi-specific antibodies.