In most cases, liquid chromatography involves using small particles or resins (also defined as media) that are packed and held in a column, also referred to as the stationary phase. These particles can be physically or chemically modified to provide specificity to bind or repel particular molecules within sometimes very complex mixtures. These columns can be run using gravity to move the solution sample, but more often they are operated at low, medium or high pressures—facilitated by mechanical pumps and sometimes specialized instrumentation to control the flow of sample through the stationary phase in the column. There is a wide array of chromatography resins available to address the evolving needs of researchers purifying a diverse set of target molecules.
Liquid chromatography can be performed at both analytical and preparative scales. With an analytical approach, the goal is typically discovery, identification and sometimes quantification of a component from a complex mixture. The focus of preparative liquid chromatography is to isolate and purify molecules typically used in downstream bioprocessing steps.
Protein purification can pose several challenges, and as with any purification scheme, protocol optimization is required. There are many options for media selection and each chromatography method serves a different purpose, whether it is a more general selection of molecules or a highly specific separation. Certain targets may require multiple separation steps using different columns, or a multi-modal or mixed resin method to address the specific needs of researchers.
Here we briefly discuss some of the more common liquid chromatography methods used for protein-purification workflows, highlighting the media (resins) and main principles and considerations of each methodology. A general rule to follow is to obtain the required purity of your target protein in the fewest steps possible.
Chromatography and media selection
Ion Exchange Chromatography
Ion exchange (IEX) chromatography separates molecules based on their total charge. This technique is a powerful method that is used at both analytic as well as preparative phases of purification. Ion exchange resins are created by covalently linking either positively or negatively charged functional groups to a solid matrix. Some commonly used matrices include cellulose, agarose, polymethacrylate, polystyrene and polyacrylamide. Protein sample is loaded onto an IEX column at low ionic strength and then washed with buffers of increasing ionic strength/salt to remove undesired proteins and impurities; the target protein(s) of interest is eluted using either defined salt gradients or a change in pH. Elution by salt gradients relies on the fact that charged salt ions compete with bound targets for the charged resin. Targets with fewer charged groups tend to elute at lower salt concentrations, and those with more charged groups are eluted at higher sal concentrations. Buffer conditions are critical to this methodology, and sample is typically loaded onto the column in low-salt condition. For certain samples, this may require a buffer-exchange step prior to loading on an IEX column. Alternatively, elution by pH exploits the isoelectric point (pI) of a target protein. When the pH presented to the sample reaches the pI of the target of interest, it no longer carries a net charge and is released by the resin.
For targets such as monoclonal antibodies, IEX is an effective second purification step; affinity chromatography (see below) is typically the initial step in the purification workflow. For non-antibody molecules, large-bead IEX resin is a good starting point for the first column purification step.
Hydrophobic Interaction Chromatograph
Hydrophobic interaction chromatography (HIC) separates proteins based on their hydrophobicity. HIC is often used to separate or purify proteins while maintaining their biological activity, as this technique utilizes buffers, matrices and parameters that are less denaturing to the sample than other purification methods. Salt concentrations, pH and temperature can all influence binding interactions with the media as well as the ligand chemistry that is immobilized to the resin. This methodology is complementary and often used in conjunction with either an upstream high-salt IEX elution or downstream size-exclusion (see below) purification procedure.
Size-exclusion chromatography (SEC) partitions proteins based on their size by filtration through a gel. The gel is made up of spherical beads containing specific-size pores that either include or exclude molecules from the pores within the media. Separation of proteins occurs by size as they pass through the column and are eluted in order of decreasing molecular weight. The two most commonly utilized SEC procedures are to fraction and desalt/buffer exchange proteins. SEC is often used to separate proteins that might not be resolved by other methods, such as IEX or HIC. Researchers may choose to reserve SEC for a final step of purification.
Affinity chromatography utilizes specific binding interactions between a ligand that is immobilized to a resin and its binding partner. The binding/purification is often highly selective and takes advantage of the target protein’s biological structure or function. Typical applications for this method include antibody/antigen, enzyme/substrate and enzyme/inhibitor interactions. Both native as well as recombinantly generated molecules can be purified by this method. Besides offering greater selectivity, affinity chromatography can potentially provide faster time-to-results because of the specific interaction. Depending on the specific purity goals, affinity chromatography is often the first step, if not the only step, in a purification workflow scheme.
Multimodal or Mixed-Mode Chromatography
Multimodal or mixed-mode chromatography utilizes resins that have been functionalized with ligands capable of multiple interactions. This method is useful when purifying target proteins that do not have a known specificity. The resin can be used to screen, purify and potentially identify sites on a target protein that can provide useful affinity and selectivity information. However, because there are multiple binding and elution properties, the target interaction cannot be predicted from simple amino acid sequence analysis and requires upfront experimentation on the binding and elution condition optimization. The main advantage of this technique is to combine complementary chromatography methods within a single medium, which can save purification steps and precious sample material while also potentially providing faster time to results, especially for product impurities that are typically very similar to the target molecule.
Many options for protein purification
Separating a target protein from complex samples can be very challenging. The purification steps are key and essential processes to further characterizing and understanding the function of the target protein. There are many variables involved in optimizing the workflow process. Often, it is best to start with the primary amino acid sequence of your target. This will provide information on the molecular weight (as a guide for SEC) and pI (as a guide for IEX) and help predict and “score” the solubility characteristics of the protein. Selecting the appropriate chromatography media, along with the wash and elution buffer conditions, can greatly assist in the purification process. There are also multimodal or mixed-mode resins for use when the properties of the target of interest are not well understood. For additional assistance, hydrophobicity plots and secondary-structure predictions can also provide guidance when considering either HIC or mixed-mode resins. Targets with disordered secondary structure (especially at the termini) are frequently either unstable or aggregate-prone, and they interact well with HIC media.
A variety of IEX, affinity, SEC, HIC and multimodal or mixed-mode resins are available from many commercial vendors. In addition, many vendors are able to provide customized media to address the individual and unique needs of researchers. Spend the time to educate yourself on the options available by using online resources and by reaching out to specific vendors to assist you in selecting and developing the appropriate liquid chromatography purification procedures and strategies.