While protein purification is a well-established, conventional and highly trusted discipline, the approaches it employs can be as diverse as its targets. This makes a lot of sense as proteins come in many different sizes, structures and possess unique binding affinities and biological activities. As such, it’s not surprising that specific proteins require purification strategies tailored to their own unique physico-chemical properties. The isolation of a mitochondrial coenzyme, for instance, will require a different approach to the purification of an antibody Fab fragment. Moreover, these examples likely represent two different scales of protein purification; the former target would most likely be purified at the exploratory level, with Fab fragments at lab scale proportions and beyond (due to their application in therapeutics). However, despite these fundamental differences in purification requirements, there are certain steps you can take towards purification success that are applicable to the majority of proteins. Here we review some essential tips for Fab protein purification.
Act quickly
You want to avoid drawn-out purification protocols that take several weeks. The temptation may be to store cells after harvesting and pelleting them, then come back to them at a later stage. But lysing cells as soon as you are able to and removing cell debris and other impurities quickly, is the best tactic for purification success. Similarly, applying your clarified cell lysate to the chromatography column at first opportunity will also produce optimal results. Moving swiftly between each of these stages isolates target proteins from elements that could denature or destroy them as efficiently as possible.
Is this step necessary?
In a similar vein, ask yourself if each step is absolutely necessary. That is not to encourage cutting corners, but there may be elements of your workflow that are just adding time to your protocol without adding very much to your final product. For instance, do you really need to remove purification tags? There are many examples where such features, especially His-tags, do not interfere with protein function or structural analyses, so why bother removing them? Removing the tag with a protease will add at least another day to your preparation. Plus, it’s always difficult to completely remove every trace of a tag, leaving heterogeneity in your sample.
Expediting buffer exchanges
A step that will save both time and unnecessary processing is the use of a desalting column over more traditional methods of dialysis. This strategy enables you to exchange buffers a lot faster e.g. in 30 minutes compared to overnight; plus, it is done in a single step as opposed to several buffer changes. You can also work it into an automated multi-dimensional chromatography set up if you have access to such resources. This would further minimize the need to handle the sample, avoiding a significant point of sample loss in your workflow.
If you still prefer to use dialysis methods for buffer exchange, make sure you opt for dialysis membranes with a Molecular Weight Cut Off (MWCO) close (but not too close) to the molecular weight of your target protein. As a rule of thumb, select a MWCO that is half the size of the MW of the species to be retained and at least 20 times larger than the MW of the species intended to pass through.
Keeping it cool
It may seem like obvious advice, but try to keep your protein sample on ice or as close to 4°C or below as possible. This goes for sample downtime; anytime your sample is not in ‘active use,’ i.e. not on the chromatography column, remember it needs to be cooled as quickly as possible. Pre-chill all your buffers before use too.
Size exclusion column
Ending any purification workflow with a size exclusion column is a smart move. Consider it an in-built quality control check. Not only does it serve as an extra purification measure, it enables you to isolate your protein in its correct oligomerization state; plus, any degraded protein is also removed. This is because protein species are eluted according to size, and not shared features like affinity or charge.
Fab fragment purification
Here are some general considerations for Fab fragment purification. As with any purification workflow, some optimization may be required.
Try E. coli
The reduced size and intrinsic characteristics of Fab proteins not only make them good choices for immunodetection, purification, and bioseparation applications, but also as therapeutic molecules. Depending on the intended application of your protein you may be concerned about expressing your protein in bacterial hosts. In a recent webinar presentation, Jianshen Wu, senior scientist shared cases studies demonstrating high yields of protein with little to no trace amounts of impurities when using E. coli hosts. We’re talking endotoxin levels below 0.1 EU/mg (providing you follow the advice further on) so if this suits your needs, why spend money and more time setting up workflows using more complex host organisms? Using a generic organism like E. coli also benefits larger scale studies that require higher amounts of starting materials. E. coli is a fast and economical way to generate large amounts of Fab.
Start with Protein G
Fab is derived from an antibody molecule with its Fc region removed, leaving a light chain connected to a cleaved form of the heavy chain (comprised of VH and CH1) via a single disulfide bond. The traditional antibody isolation protocol using Protein A affinity capture chromatography relies on the capture of the Fc region. Obviously, this approach is not suitable for isolating Fab, and it’s also a good idea to stay clear of trying to use target affinity for this purpose too – you don’t want to risk your Fab molecule binding too tightly to the purification resin, or damaging antigen binding sites trying to elute them using harsh protocols. So which purification medium is best?
A common option is using a Protein G-based resin; although the remaining CH1 domain doesn’t interact as strongly with Protein G as the CH2 and CH3 domains, there is still a certain degree of attraction. This interaction also happens regardless of isotype, and binding is seen in the case of both Human and mouse Fabs. This makes Protein G-based resins a good starting point for any Fab purification protocol as it works across the board. It is also a good strategy, if using Protein G, to reapply flowthrough to the column at least once to several times more, as this should increase the yield of Fab protein.
Understand binding capacity
The term “binding capacity” is somewhat vague because manufacturers tend to mean different things by it. It can refer to “breakthrough capacity”, which only reflects what you would see if the material on the column was only the molecule of interest. “Dynamic capacity” is also used to qualify chromatography media, and this gives a more accurate idea of performance as it relates to the maximum quantity of loaded protein that will still allow recovery of pure product, but it is often significantly less than breakthrough capacity. It is also less reproducible than the latter and hence why breakthrough capacity is the measurement preferred by manufacturers.
There is another measurement of binding capacity called “apparent binding capacity,” which is even more accurate than dynamic capacity. It requires multiple experiments from the user to calculate, but it helps you to confirm whether you are using the right size of column.
Acceptable recovery rates fall between 60-80%; in this range there is a good match between the apparent binding capacity and column size, and it is in this range that separations are most efficient. However, there are exceptions to this; in the case of such outliers, a higher load does not lead to a higher binding capacity, and these examples may signal low affinity binders.
Try alternatives
If you encounter a low affinity binder—for instance you only obtained around 15 percent of target Fab protein from your lysate—consider using an alternative chromatography resin. Capture media that specifically target the Fab domain are an option, as are resins made up of modified versions of Protein G. It is worth experimenting with these media as some retain certain Fab fragments better than others.
Use these steps to increase purity
With the right combination of buffers you can ensure your E. coli-expressed Fab fragments are eluted from the chromatography column with little to no trace of endotoxins, and in the correct oligomerization state. Washing with a buffer containing Triton-X 114 helps remove endotoxin, while adding a glutathione (GSH)-based washing step reduces the amount of cysteine adducts between fragments, thus reducing any Fab2 present. Adding a secondary purification step after these washes, such as ion exchange, or even a third step (see the point on size exclusion above) will increase purity even more. This was demonstrated by Jiansheng Wu, senior scientist from Genentech, in a recent presentation. He shared that using Triton-X 114 and GSH washes through a modified Protein G column, before further purification on a cation exchange column, 4.1g of target Fab was successfully purified from 700g of cell pellet, with below 0.1Eu/mg of endotoxin and 98% monomeric Fab.
Concluding remarks
The increasing use of Fab fragments in both research and the clinic, mean large batches of incredibly pure Fab proteins are routinely required by a growing number of laboratories. Because of this, Fab purification workflows need to be cost effective, streamlined and boast a high degree of universality. By following the advice above you should attain high yields of pure Fab protein, in taking measures that uphold the aforesaid parameters.
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