Bench Tip: Lab Scale Protein Purification

Abstract

Protein purification at the lab scale bridges the gap between small-scale exploratory protein purification and high-throughput operations, such as industrial-scale manufacturing. At the larger end of the scale, advances in upstream processing such as improved fermentation capacities have led to increased amounts of crude sample available for input. 

Despite this meaning higher potential yields of target proteins, it also poses an enormous challenge for timely and cost effective sample processing further downstream. This challenge is made even more difficult as scale increases due to higher amounts of impurities – a result of prolonged fermentation times and higher cell densities in large-scale cell cultures.

In general, a lot of optimization work is done at the initial, exploratory protein purification phase. Here, you’ll have identified whether your target protein suffers any solubility issues, which buffers it’s happiest in, and a basic strategy for its purification. The work carried out at the lab scale stage , however, is crucial for making a successful transition between exploratory and large-scale protein purification. It enables you to investigate how to increase your working volume, without compromising on product recovery or purity.

Typically, many protein purification protocols rely on the application of chromatography, no matter the volume being handled. Lab scale procedures are no exception to this trend, and here the chromatographic process can be broken down into three main stages: target protein capture, intermediate purification and final polishing [1]. Each of these stages performs a specific function in the processing workflow, and as such has its own unique set of points for consideration when it comes to process design. Similarly, each stage presents different challenges during scale-up. This article discusses important factors to consider when developing a downstream workflow for lab scale protein purification with a focus on chromatography.

Protein Capture

The basic aim of protein capture is to collect and retain as much of the target protein as possible, as quickly as possible, while letting unwanted impurities flow unhindered through the separation medium. At this stage, the resolving properties of your chosen chromatography resin is not as important as its binding capacity for your target protein.

Affinity chromatography resins are often employed at this stage of protein purification. They are an efficient method of target protein enrichment, quickly sequestering them from any impurities that could potentially damage them. They work on the basis of a protein’s attraction for a matrix-bound ligand, and this interaction may either be mediated by native protein moieties or by the addition of an affinity tag by engineering. If your process involves an exogenous tag, think carefully how you will remove it at the end of the purification procedure. First ask if you need to remove the tag at all; tag removal by protease treatment could add at least another day to your protocol. If it is an absolute must, remember to build purification steps into your downstream workflow that will remove the tag entirely, so as to avoid heterogeneity in your final sample (e.g. add an uncleavable version of the tag to your protease and run the sample through the affinity column again to capture the protease, the cleaved tags, and any uncleaved target protein; this leaves tag-free protein to flow through the column for easy separation ).

If you need to avoid using affinity based purification as you wish to avoid introducing a tag to your target protein, ion exchange chromatography resins could be another option for protein isolation. These are discussed in greater detail below.

Whichever capture method you opt for, consider that this initial stage will process the largest sample input volume; therefore, a relatively fast flow rate will be required to preserve the efficiency of your workflow. Opt for a larger bead resin possessing good flow characteristics (for example, a very hydrophilic bead material) to improve overall processing times without compromising on binding capacity. This will work in your favor when the process is eventually scaled up.

Intermediate purification

Resolution of your target protein is a bigger priority at the intermediate purification stage. Some host cell contaminants will remain lurking in your sample, even though the bulk of impurities will have been removed in the previous step. It may be tempting at this stage to use chromatography resins with the finest bead size possible as bead size correlates with final product resolution; However, medium-sized beads are often more appropriate for this stage of the process. Speed is less of a concern, as any impurities that would affect the stability or activity of the target protein will have been removed in the capture step. But still, medium bead sizes will offer the best compromise between processing times and purity as they still allow for the removal of heterogeneous elements such as charge variants or truncated proteins.

Ion exchange chromatography is a popular option for intermediate-stage purification. It captures proteins based on their charge and their attraction for an oppositely charged matrix. Separation can be carried out by using increasing gradients of ionic strength or pH, or isocratic elution, where the mobile phase remains unchanged but promotes faster or slower passage of species though the matrix based on polarity. These differential approaches to protein separation sees target proteins enter back into the mobile phase during distinct elution windows, thus allowing fine-tuning of elution and further concentration the target protein.

You should also consider using a mixed-mode resin at this intermediate stage. Mixed-mode resins offer unique separation properties due to their composition which is comprised of multiple separation modalities. Increasing the number and variety of separation methods used in the purification process results in higher-quality end products. Using mixed-mode resins streamlines this tactic and provides unparalleled selectivity and resolution for a wide range of biological molecules. It also enables sample loading at higher salt concentrations, depending on the target protein and mixed-mode resin used. This eliminates the need for buffer exchange or dialysis steps that are often required for ion exchange chromatography, reducing the overall processing time and potential for sample loss. The use of mixed-mode resins is also applicable to the final polishing step.

Final Polishing

In the final polishing stage of chromatography, the main goal is to achieve a high resolution of your target protein by removal of trace contaminants and also isolate it from any unwanted structural variants. A small bead size is also recommended for final polishing chromatography. Note that the use of a smaller bead size will result in increased pressure; in this case, lower flow rates should be used thus meaning process times can be longer.

Size exclusion chromatography (SEC) is often used for this purpose as it removes any lingering impurities, but also deals with chemically similar, but structurally unsuitable protein species, such as dimers or degraded versions of your target protein. It is a nonbinding technique that, as the name implies, separates proteins based on their size differences. Isocratic buffers are used as standard, and it is important to consider that SEC matrices can only handle low sample volume loading (on average ≤10% of matrix volume) if you want to achieve optimal resolution.

Final word

Lab scale protein purification lays the foundations for future processing success, so it pays to design a workflow that not only results in the purest protein product, but also remains scalable for the future. Consider the use of the chromatography resins in the order outlined above in your lab scale experimentation. Remember that a variety of resins are available within each media category from different vendors, so it would also be beneficial to consider the pros and cons of each in your design process. Some are better suited to the demands of lab scale processes than others.

References

1. Milne JJ, “Scale-up of protein purification: downstream processing issues,” Methods Mol Biol. 681:73-85, 2011. [PMID: 20978961]