Once protein purification work surpasses a certain scale in terms of volume, a measured and step-wise development of the bioseparation process is vital, keeping in mind possible further expansion of the workflow process farther along the lifespan of the purified product. This covers volumes of one liter all the way up to 2,000 liters and even beyond, where the separation process is referred to as downstream processing. Just as with exploratory and pilot scale protein purifications, downstream processing can be broken down into four consecutive stages: removal of insoluble materials, product isolation, product purification and product polishing. Of these, some steps offer greater opportunity to refine workflows over others, but any optimization work essentially focuses on how to get the most out of downstream processing for the lowest cost. The gains sought after here, however, are not just in terms of monetary savings and product yield; most importantly they also seek to maintain or even enhance product purity as, often, this scale is employed for the manufacture of protein-based therapeutics. This article provides tips on how to get the most out of your downstream processing workflow as well as considerations for its initial set up.
Start early
There is perhaps one stage of bioseparation that gets a little overlooked in the grand scheme of downstream processing. And that’s because it actually comes before you even get near any separation apparatus, at the cell culture growth stage. Failure to investigate the changing parameters of large-scale cell culture can spell disaster as you progress into downstream processing – even if you have optimized this stage previously during upstream processing. Larger bioreactors, increased culture volumes, higher oxygen demands and greater amounts of waste products can all contribute to changes in the expression of your target product. Such changes could include, for example, protein glycosylation patterns, which would lead to problems with protein-matrix interactions during product purification later on. They could also alter the contamination profile of your product, challenging product purity further downstream. Furthermore, successful fine-tuning of cell culture conditions could significantly increase target protein expression and, thus, end-product yield. Therefore, it pays to examine your workflow at the cell culture level before optimizing anything else. Adjustments here can set you up for success further down the line; essentially, the more you put into downstream processing, the more you should get out of it at the end.
Keep it simple …but not too simple!
It may seem like contradictory advice, but keeping your downstream processing steps as simple as possible will work in its favor. Designing the process workflow so that feed streams can be added directly to subsequent steps without the need for buffer exchanges or dilutions, ideally as part of a closed system, will increase its efficiency and should lead to higher product yield as a result. Keeping the number of steps required to a minimum will also benefit your procedure in the long run.
If you’re worried that streamlining the process in this way will compromise product quality in any way, the advice above is not to say that you should set aside any sort of orthogonal approach. But it’s possible to obtain the levels of purity required of protein therapeutics with only two subsequent chromatography columns, if you select each resin considering the process as a whole. The domain of chromatography media has recently witnessed several innovations in terms of resin composition; moving towards universal applicability in terms of scale and facilitating the development of closed, fully integrated systems.
Consider mixing it up
Mixed mode resins are gaining popularity with process developers as they enable researchers more flexibility in workflow design, without sacrificing product purity. These resins combine the properties of two orthogonal interactions, such as cation exchange and hydrophobic interaction. These properties make the separation of biomolecules, even closely-related ones, much less problematic. Moreover, they can reduce the number of steps required further downstream, thereby decreasing product loss and lowering costs – two benefits advantageous to any large-scale process.
Several biotech companies offer mixed mode resins, presenting you with a range of options. One such solution is Nuvia™ cPrime™ developed by Bio-Rad to facilitate versatile capture and high recovery of target proteins across a wide range of salt concentrations and pH. This robust tolerance for target binding and retention under variable conditions can enable the direct loading of the feed stream onto Nuvia cPrime columns without the need for any pre-dilution. This cuts down on processing time, thus increasing efficiency and adding another benefit to using mixed mode chromatography media.
Give it a good polish
The final step in the purification workflow is typically a polishing step. During this process, any contaminants or undesirable forms of the target protein are removed from the preparation.
When selecting resins for this important step, keep in mind the buffer composition you are likely to use to elute your product from the previous chromatography column. Choosing a polishing resin that will bind to your target product in this buffer will spare you the complications of having to work a buffer exchange step into your workflow, saving you time and money, and mitigating product loss. In addition to commonly used cation exchange columns, mixed-mode resins such as CHT™ ceramic hydroxyapatite, are another great option for researchers due in part to their unique selectivities and diverse binding capabilities. Vendors specializing in chromatography products, such as Bio-Rad , have large inventories of resins to choose from and can offer expert advice to help customers looking for a suitably compatible polishing resin or even a custom-tailored solution for their process.
Quality control
All protein products intended for clinical use require adequate monitoring for quantity and purity. And not just at the end of the process; online processing should be integrated into any downstream processing workflow where feasible. This means being able to sample the feed stream at appropriate points and test these samples as rapidly as possible. A means of host-cell protein (HCP) detection must be included in your test kit, and there are several ways this can be done from simple ELISA testing to Western blotting for specific HCPs. If you opt for the latter method, Bio-Rad provides ready-made workflow solution optimized to provide consistent protein separations, and high-sensitivity, reliable detection on Western blots
Alternatively, there are biotechnology companies that specialize in providing HCP solutions for larger scale processein which the HCP detection antibody panel is tailored to the exact process (i.e. raised from the contamination profile of your chosen host cell, when grown under consistent conditions). Using such services also helps take the hassle out of meeting the HCP testing requirement.
Product purity also extends to the product itself – has the protein aggregated or degraded? In this regard, immunological assays do not provide enough detail, so here, analysis using methods such as size exclusion chromatography should be considered. Using a chromatography set-up capable of measuring protein concentration at an absorbance of 280 nm will help you monitor protein concentration online, but because this is vulnerable to errors – depending on buffer components or the presence of impurities – it’s always smart to use a dye-based assay, such as a bicinchonic acid (BCA)-based one, if not a Bradford/Lowry assay, to confirm protein concentration after the final purification step.
Summing up
Each downstream processing protocol will vary depending on the molecule targeted for purification, but if you follow a logical, step-wise development, taking into account all process components, you will see your process succeed. Reach out to the technical support team from your preferred chromatography system and resin provider as they have a wealth of experience in both upstream and downstream processing steps and can provide guidance to get you through the trickiest steps.
Image: Dreamstime Images
Related Products from: Bio-Rad