In this section, you will find audience questions and thorough answers from our application scientists grouped by common chromatography topics.
Sample Preparation
How are sample lysates usually prepared before applied to the column, filtration, centrifugation, etc., and how does the scale affect this procedure?
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A chromatography column is essentially the most expensive filter you will ever buy. You will always need to remove any aggregates or solid materials from your lysate before loading it on your column. If you are working with a biopolymer, make sure there is nothing in your buffer that will initiate polymerization while on your chromatography system. Cells are typically resuspended in a lysis buffer, on ice, and homogenized mechanically. The addition of a protease inhibitor and nucleases can help in preventing the degradation of your protein while degrading genomic material. Once homogenized, the cells can be lysed by sonication, a French press, or a microfluidizer.
Regardless of the scale of the solution you are preparing, you will clarify through centrifugation and subsequent filtration. Multiple rounds of centrifugation may be necessary. If you are afraid of disturbing the pellet by pouring off the supernatant, use a serological pipet instead. Choose filters that don't bind protein — avoid nylon and cellulose. The cleaner your lysate is going on your column, the less cleaning and regeneration will be needed, leading to longer column lifetime. As you increase in scale to manufacturing quantities, it is common to express your proteins such that they are excreted into the media so that lysis and centrifugation aren't needed. In this case, tangential flow filtration is used to concentrate the excreted proteins before chromatography.
I am having problems with protein precipitation. What are your recommendations for aiding in solubility?
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Since the standard 6X-His tag disfavors the solubility of proteins, the addition of this tag can sometimes cause proteins to precipitate. You can incorporate a solubility tag between the His-tag and your protein of interest. Some other more soluble tag options other than 6x-His include Maltose binding protein or GST-tag. You can make these cleavable by adding a cleavage site like TEV. Make sure all necessary cofactors are available for your protein during expression and are not inadvertently stripped away by incompatible buffer additives.
Sometimes, proteins have a binding partner that greatly increases stability; you might have to co-express with such a cofactor for stability. You can also add 20% sucrose for high osmolytes. Also, don't forget about the pH of your buffers! Confirm your pH with a meter at the temperature you plan on using your buffer. The pH of your buffer cannot be equal to the PI of your protein. Proteins like to be charged and not isolated!
Chromatography Methods
What are some of your suggestions to help me increase yield and purity?
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Get to know your protein's properties: pI, hydrophobicity, molecular weight, cofactors, and binding partners. Select a high capacity column for your first purification capture step. Try screening several different column types of the same chemistry. Your proteins don't only interact with the functional groups on the resin, they also interact with the base beads. Ensure the buffer system you choose won't interfere with your column chemistry.
You may want to do trials with your purification methods by using step or linear gradients. With a linear gradient, you can reduce the slope to improve your overall purity. Slowing the speed of your sample loading on the column can also help increase your yield. When expressing your protein, search for an optimal temperature for induction. The addition of glycerol during expression can be helpful in some instances as well.
Do you have any tips for peptide purification rather than protein purification?
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Adding a solubility tag to a peptide will do you a lot of good. You can also consider a denaturing purification utilizing 6–8M urea. If your peptide is too hydrophobic for typical protein chromatography techniques, you might need to use reversed-phase HPLC. If you have a multi-wavelength detector use 215 nm to monitor the peptide backbone. You can also reduce the bead size and pore size of your resin to prevent larger proteins from interacting with the resin.
Is Expanded Bed Chromatography still being used in industry?
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Expanded bed chromatography is not commonly used by our partners in the Biopharma industry. Continuous chromatography has become the preferred platform for most manufacturing sites, allowing for columns to be cleaned and regenerated simultaneously as additional columns on the system are being used for protein purification.
Do you have any experience with twin-column (tandem) chromatography?
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With two column valves, make sure the columns are compatible with both the pressure and the buffer. Optimization is needed, but it minimizes the hands-on time. The less manual intervention, the more efficient your purifications will be. If the first column elutes with a step gradient, this makes the process much easier. See our tech guide for how to use two SEC columns to increase resolution.
I am working on a new protein target. Where do I start? Are there any columns or resins would you recommend?
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Any time you start with a new protein, you need to do literature research to see what strategies have been used successfully before. Expasy is a wonderful online resource to learn about your protein properties based on its sequence. Adding an affinity tag to your protein and using an affinity capture resin is always a good place to start. Create it with a cleavable linker for downstream tag removal. Look at the pI of the protein to determine what type of ion exchange is acceptable for your protein and the pH of your buffer you should use. For smaller proteins, choose a resin with a smaller pore size. For larger proteins, chose a resin with a lager pore size. Nuvia HPQ is a good fit for large protein complexes.
Do you recommend a "polish" purification before standard purification / exchange? If so, what would this look like?
Polishing steps are typically used as the final purification step to get the cleanest and most homogeneous sample possible. The workflow typically starts with capture, followed by intermediate purification, and polishing as the final step if necessary. Take a look at our lab-scale resin selection guide on page. 3 for a nice visual representation. Polishing columns are typically size exclusion, high-resolution ion exchange, and mixed-mode resins.
Product documentation references:
When should I choose chromatography compared to capillary electrophoresis (CE) for protein purification?
Chromatography should be your technique of choice if you need to produce large amounts of very pure protein that you can use in your downstream experiments. By having the choice of many different columns of many different sizes, you have a large amount of control in your method optimizations. Also, if you have a chromatography system with a Multi-wavelength detector, you can monitor not only the 280 nm absorbance but also other protein properties such as cofactors or recombinant fluorescent labels.
What solvents are compatible with my chromatography system?
Take a look at our NGC System chemical compatibility document! If you are using another machine, refer to the manufacturer's instructions.
Product documentation references:
Chromatography System Maintenance
What maintenance should I be performing on my instrument?
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Look over your machine for drips and air in lines before every use. At the end of the day, flush out all of the buffers from your system with water, followed by 20% ethanol to keep the flow path sterile. Be sure to flush through your fraction collector as well. If you have a sample pump, clean the pump with 0.5 M sodium hydroxide after every use. Always bookend ethanol with water to prevent salt precipitation. Change out the 20% ethanol pump-head wash solution and manually prime the lines with a syringe weekly. Calibrate your pH probe every day. Reboot your machine and computer weekly. Check your inline filters for clogs. Clean your columns following manufacturer's recommendations, and preferably in reverse flow, if allowed. Perform annual preventative maintenance including a check of your system pumps, and replace the piston seals.
I left the lab in a hurry to shelter-in-place, what would you recommend I do to my system before I use it again?
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Start with a thorough visual inspection. Reboot your system and your computer. Look into all of your buffers and inlet lines for any microbial growth. Make all new buffers, which are filtered and degassed. Run your system with water and check for blockages or leaks with no columns in line. You can use water warmed to 40ºC for extra solvation power. If you find a clogged piece of tubing, you can use a syringe with warm water to flush or sonicate the tube. You can use one of our clean-in-place protocols. If you want to ensure your system is fully sanitized, follow our sanitization protocol in the resource section.
Product documentation references:
Any tips for cleaning clogged columns?
Look to the manufacturer's instructions on cleaning procedures. Typically, they will have multiple cleaning and regeneration suggestions depending on how thoroughly your column needs to be cleaned. If your column allows for flowing in reverse, you should definitely clean it bottom to top. This will prevent all of the junk that may have gotten stuck on the top frit from getting pushed further into your column.
How long can I keep the gel filtration column in running buffer instead of 20% EtOH until the next run?
The general rule of thumb for your chromatography column is that you can leave the column in a buffer if you are planning on using it again within the next 24 hours. If you will not be using it again within that time frame, wash it with water and take it to 20% EtOH for storage.
Coronavirus Research
How is chromatography utilized in the development and manufacturing of vaccines?
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Chromatography is heavily used in vaccine manufacturing as the final steps to purify the components that go into a vaccine. The material used in the vaccine must be completely pure and homogenous. Affinity tags cannot be added to the proteins, so separation based on the inherent properties of the molecule are done through ion exchange and mixed-mode resins like CHT.
How do researchers utilize protein chromatography to study novel viruses like MERS, SARS, and now SARS-CoV-2?
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Researchers are actively studying the spike protein complex of the SARS-CoV-2 virus and the human ACE-2 receptor to which the virus binds. Researchers overexpress and purify these individual components to conduct biophysical and structural studies of these interactions. The specifics of these interactions allow researchers to create and further investigate the efficacy of rationally designed therapeutics.
Miscellaneous Chromatography
What types of resins do you think undergraduate students should learn about/use in teaching labs to get them ready for biomedical research? Or, what are the most commonly used resins used in biomedical research?
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Students need to understand the chemistries of the resins and how they are used in typical purification workflows. Affinity capture, ion exchange, and mixed-mode resins are the most relevant to today's biomanufacturing landscape. Size exclusion and desalting are other techniques to teach your students but aren't regularly used outside of R&D. As for the flow of learning, we would suggest starting with affinity, then ion exchange, and then mixed mode. Teach them to consider how the media bead size, hydrophobicity, and porosity can influence your protein's interaction on the column.
Mixed-mode resins are used widely in the manufacturing world and are starting to be adopted in research labs as well. Using mixed-mode resins requires more up-front optimization, but is a very efficient process in the large-scale manufacturing world due to their ability to simultaneously exploit multiple types of interactions with the column resin relying only on the protein's native properties.
You all mentioned a lot about resin chromatography? Do you offer any porous membrane for chromatography?
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We do not currently offer chromatography membranes.
