Protein Purification Products & Processes

Most commercially available protein purification kits use some form of column chromatography―often in the form of packed agarose beads. Biocompare recently interviewed three companies to learn about what advances they are making in the field of protein purification. While some advances involve new purification techniques, others involve emerging strategies to assess protein purity.

From agarose resins to magnetic beads and fibers

Most commercial protein purification kits use some form of affinity chromatography—in which the protein of interest is purified due to specific binding with an immobilized ligand. These ligands have traditionally been attached to porous agarose beads to act as the stationary phase in chromatography. However, agarose beads have been associated with diffusional and flow bottlenecks.

In recent years, magnetic beads have replaced agarose beads in many applications. “The technology for magnetic beads isn’t brand new, but magnetic beads for protein purification have been a fast-growing market,” Cynthia Chen, Product Manager for the Protein Prep & Reagents division at MilliporeSigma, explains. “They allow for automation, which makes it easier and faster to purify recombinant proteins.”

In contrast to agarose beads, which often require time-consuming centrifugation steps, magnetic beads can be isolated with a magnet―either by hand or with an automated platform. This leads to more efficient purification of the sample. Last year, MilliporeSigma launched additional lines of magnetic beads, including anti-HA, anti-c-Myc, and anti-V5 beads. “This year, we’ve got a few more in the works to build out our product offerings for different protein tags,” she adds.

Henrik Ihre, Business Leader of Next Generation Products at Cytiva, notes that in addition to conventional agarose beads, Cytiva has developed new fiber-based technologies. “This technology is suitable for recombinant proteins and for really large target molecules such as viral vectors, plasmids, and mRNA,” he says.

Fiber chromatography is a separation technique that uses cellulose fibers. And while conventional chromatography relies on the slow diffusion of molecules through the pores of a bead, fiber chromatography is based on convective flow. This allows for residence times that are seconds rather than minutes in the case of agarose resins.

What can go wrong?

Another key question for researchers involves assessing protein purity once the purification process is finished. Soeren Rowold, Area Sales Manager for Implen, explains that, prior to protein purification, cell lysates often contain many contaminants such as cell debris, nucleic acids, fatty acids, polysaccharides, and other proteins that are not of interest. “All these substances need to be removed,” he warns. “Additionally, the efficiency of downstream applications like blotting might be affected by certain contaminations.”

Chen notes that several things tend to go wrong with protein purification. Sometimes, protein tags, which are often attached to recombinant proteins, may not be adequately expressed. For instance, the protein tag may be hidden by the protein’s structure, preventing it from binding to the affinity resins. In other cases, wash buffers may be too harsh, especially if the pH is off or if chelating agents degrade proteins. Finally, the sample may be too crude. “Sometimes the protein is in a really concentrated sample, which throws off the balance of required washing and affinity resin volume,” Chen explains.

Ihre says that it may be necessary to carefully follow yields and purity during the different steps of the purification process. “Purity and yield are often contradictory, so that is why it is important to balance the two parameters to achieve the best possible overall result,” he says. Furthermore, he notes that both design of experiment (DoE) and mechanistic modeling tools can be harnessed to design an optimized process for purity and yield.

Assessing protein purity and structure

Ihre and Chen suggest various techniques to assess the purity of an isolated protein―including enzyme-linked immunosorbent assay (ELISA), size-exclusion chromatography (SEC), mass spectrometry, high-performance liquid chromatography (HPLC), and electrophoresis. In particular, Chen recommends performing a western blot after gel electrophoresis using an antibody probe that recognizes the protein of interest. “For all of these methods, not only would you be looking at the intensity of a signal―whether it is a band on a gel or a chromatography peak―but you’d also want low background, which would indicate how pure the sample is,” says Chen.

Rowold notes that a droplet spectrophotometer, like the Implen NanoPhotometer, is another useful way to assess purity. “Proteins obtained from lysates are often contaminated with DNA. The A260/A280 ratio is a valuable parameter to assess the purity of isolated proteins,” he says. He also emphasizes that the ideal A260/A280 ratio for typical proteins is less than 0.7. “An elevated ratio may suggest the presence of DNA in the isolated protein sample,” he warns.

Finally, Ihre advises that, “It is a good idea to look at protein stability to make sure that your protein isn’t being altered in some way during the purification process.” He notes that surface plasmon resonance (SPR) reflectivity measurements, which analyze changes in the local index of refraction upon adsorption of the target molecule to a metal surface, are increasingly being used to assess protein structure and biological activity. In particular, he recommends Cytiva’s SPR-based Biacore technology.

What level of purity should we aim for?

Ihre notes that different levels of protein purity may be required depending on the application and the field. “From an academic and scientific perspective, a highly pure protein can more readily be studied from a structural and biological perspective. However, from a therapeutic perspective, a protein needs to be highly pure to ensure patient safety and to get market approval by regulatory authorities.”

Ihre further emphasizes that the desired purity level may depend on the intended use of the protein―with current applications ranging from medicine to food to agriculture. Nevertheless, he notes, “For many applications, a purity above 99% is desired or even required.”