Protein Purification Support—Getting Started - Thermo Fisher Scientific

The benefits include high-capacity immobilization of 1–10 mg of oxidized antibody or other glycoprotein per milliliter of resin. Immobilization is fast (in as few as 30 mins) and can achieve at least 90% coupling of most glycoproteins in less than 4 hours. The hydrazide-activated UltraLink™ resin conjugates only to purified glycoproteins whose sugar groups have been gently oxidized with periodate.

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No stabilization step is required when using this resin, and antibody function is preserved as IgGs are immobilized via the Fc region, keeping both antigen binding sites available for capturing targets.

Affinity chromatography uses a ligand that is coupled to a solid support. When a complex mixture is passed over the column, only those molecules having specific binding affinity to the ligand are bound and purified.

Affinity chromatography can be based on either positive selection or negative selection. This depends on what is bound to the column and the recovery method for the target of interest.

In Positive Selection, a ligand that will be the specific molecule(s) of interest is immobilized as bait to pull out the target molecule(s). Following the binding step, the non-bound molecules in the mixture are washed away, and the target protein is recovered using an elution buffer that releases the target from the bait. Elution can be performed by any means that will cause the bait molecule to release the target molecule. Common elution buffers are those that result in a change in pH, salt content, or otherwise result in a change of the three-dimensional structure of the bait and/or target, resulting in its release. Displacement by a small molecule that is structurally similar, such as glutathione for glutathione- s-transferase, is also a common elution method. Such displacement methods of elution are often more gentle and can result in a more functional target molecule upon recovery of the target from the affinity support. The majority of affinity chromatography is based on positive selection. To learn more about typical elution options for affinity purification, please refer to this Tech Tip.

In Negative Selection, a ligand is immobilized in order to bind and remove contaminating component(s) in the sample. Therefore, you eliminate what you do not want in your sample in order to keep what you need in a usable form. The use of negative selection protects the molecule of interest from exposure to elution conditions preventing possible denaturation or other damage, and reduction or elimination of downstream clean-up steps.

For general affinity purification or affinity chromatography of proteins, certain affinity pairs are recognized to work well for purification of a target molecule.

Common protein ligands include:

• Charged metal ions reactive with specific amino acids (e.g., Ni++/His purification)
• Other proteins with specific affinity to a natural or artificial feature of the target protein (streptavidin/biotin)
• Antibodies (immunoprecipitation)
• Fusion tags; small amino acids added to a target to allow for ease of purification. Fusion tags are often designed so that they can be readily removed from the target following purification.

Batch method purification can be performed at any scale, however it is most commonly reserved for microcentrifuge tube–scale purifications involving 10–200 µL of resin. In batch method purification, wash and elution fractions are separated from the resin after centrifuging to pellet the resin beads. The liquid cannot be removed completely because some of it is contained within the volume of porous bead pellet. Consequently, a portion of each fraction about equal to the volume of resin used is left behind in the pellet, making washes and elution somewhat inefficient.

The spin cup purification method provides improved efficiency of wash and elution steps relative to the batch method. Centrifugation separates the liquid fraction by withdrawing it thoroughly from the resin, which is retained within the spin cup apparatus. Spin cup purification is most appropriate when 50–300 μL of immobilized ligand resin is used.

Read more about these purification methods in this Tech Tip.

CaptureSelect™ affinity resins can make the purification of biomolecules that do not have a traditional affinity purification solution much more efficient. When a targeted, specific affinity purification solution does not exist for a biomolecule, the protein purification scheme can be very complex needing 4–5 chromatography steps. Many chromatographic steps need to be utilized to separate the product of interest from key process and product related impurities. As the number of required purification unit operations increases, the product yield decreases and is heavily impacted with each step added. Yield drives cost of goods, so not having an affinity purification solution can greatly impact the cost for biotherapeutic manufacturing. Using in-house capabilities and expertise, CaptureSelect™ Custom Services design product-specific affinity ligand based on single-chain, camelid-derived antibodies, and couple that ligand to a high-performance, cost-effective affinity resin. We currently offer off-the-shelf bioprocess resins for antibody fragments, biosimilars, and viral vectors. Custom ligands can be used for biomolecule purification, scavenging of challenging impurities, and quantitation or detection of biomolecules. Custom resins are suitable for use in a cGMP production process.

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Increased titers and product demand have caused substantial bottlenecks in downstream processing for a range of biomolecules. POROS™ chromatography resins address these challenges with solutions that help maintain performance and add process flexibility. Due to the polymeric nature of the backbone and the way we attach the ligands, the beads have very robust physical and chemical stability. This allows for aggressive solutions to be used, if needed, to improve cleanability and thereby increase resin lifetime/reuse. Our ion exchange resins are the go-to resins in downstream monoclonal antibody and recombinant protein chromatography where capacity, resolution, and yield are critical. We are also working on a new line of hydrophobic interaction resins that may be of interest for future projects that will be a best-in-class product for capacity and resolution with an added benefit of being able to load under lower salt concentrations and faster flow rates. We are also a strong partner when it comes to quality and supply of our products.

Resin lifetime depends on how the resin is used and the cleaning process that is employed. Therefore, each purification process will need to be evaluated specifically, especially if long lifetime is desired. Because binding can be different between resins, different cleaning schemes may be needed. An unoptimized cleaning process of any resin may yield <5 uses, and an optimized cleaning process can yield a hundred cycles or more. Discoloration of resin can occur for different reasons from process compounds such as metals like iron (Fe2+/3+) and copper (Cu2+), additives to the media (like vitamin B12 and folic acid), and elution solutions (like magnesium chloride, Mg2+). We can provide recommendations that have proved helpful for many customers to optimize the life of their POROS™ or CaptureSelect™ column.

In general POROS™ resins have behaved better for large biomolecules such as viral particles, fusion proteins, and globular proteins, where shape (not size) matters due to its unique pore structure and large pore structure as compared to traditional resins. As the target molecule size increases, capacities obtained will decrease. The large pore structure of POROS™ resins, which allows for convective flow (and therefore enhanced diffusion) is especially well suited for the purification of large biomolecules. POROS™ resins offer the best blend of high capacity (associated with chromatography beads) and improved chromatography efficiency (typically associated with monoliths or membranes). The average pore size for POROS™ resins is 1,000–3,600 Angstrom (100–360 nm) depending on the base bead/chemistry being utilized.

For example, the molecular weight of IgG is 150 kDa and the molecular radius is 55 Angstrom. The molecular weight of IgM is ~900 kDa (pentamer), and the molecular radius is 120 Angstrom. So both of these biomolecules can interact with the pore structure associated with POROS™ materials.

UPLC is typically described as chromatography with sub-2 µm particles, which drives higher efficiency/better separation independent of operating flow rate. As particle size decreases, backpressure increases so the UPLC systems tend to have higher pressure ratings compared to HPLC systems. The flow rates on UPLC systems would be sufficient for POROS™ columns. Pump capabilities are typically 0.1–1.0 mL/min, so not as fast as a HPLC, but sufficient. Hold-up volumes on UPLC systems are lower than HPLCs, so this can help with sensitivity and efficiency. So from a pressure and flow standpoint there should be no challenge to operate a POROS™ CaptureSelect™ column on a UPLC system.

That being said, care should be taken running POROS™ columns on UPLC systems. Higher pressure safety interlock MUST be set at the rated column pressure, typically 180 bar. HPLC column hardware is not rated to operate at the static pressures possible and typical on UPLC systems. A clog or the like on a UPLC system could generate unsafe pressures on any HPLC-rated column before the default UPLC safety pressure interlock is tripped.

Fusion tags are pieces of proteins (such as glutathione S-transferase (GST)) or amino acid sequences (such as His-Tag, c- Myc tag, or HA tag) that can be added to a protein expressed in a cultured cell in vitro and can be easily detected or purified. The resulting proteins are referred to as recombinant proteins as the DNA for the original protein has been recombined with the DNA of the fusion tag in order to produce one protein that has sequences of both the original protein and the fusion tag. Adding a tag to a protein can give it a specific binding affinity.

Fusion proteins (recombinant proteins with fusion tags) can be produced (expressed) in prokaryotic cells, such as E. coli or eukaryotic cells, such as mammalian cells. Fusion proteins expressed in eukaryotes may be glycosylated or otherwise posttranslationally modified. The systems for these modifications are typically missing in prokaryotic cells.

When purifying His-tagged proteins proteins from E. coli lysates, keep in mind that there is a 29 kDa endogenous protein SlyD. SlyD has a histidine-rich c-terminus and is found in all strains of E. coli and Salmonella. The contamination is apparent when the His-tagged protein is expressed at a low level or not expressed at all. In such cases, SlyD will bind to the nickel column with great affinity. Increase the purification stringency to overcome SlyD binding.

If protein is released into LB media from E. coli, try native isolation conditions. Dialyze against binding buffer and possibly concentrate before going on to the ProBond™ resin (10% glycerol in the dialysis binding buffer will concentrate the secreted protein well). Another option is to add about 24 g NaCl and 2.8 g Na2HPO4 per liter of media, and adjust the pH to 7.8 with NaOH or HCl. This will turn the media into pseudo-binding buffer (~500 mM NaCl, ~20 mM NaPO4, pH 7.8); perform binding, washing, and eluting with either imidazole or by altering pH.

Alcohol oxidase (AOX protein) is an octamer and has at least a few His stretches. Hence, AOX protein will bind to the ProBond™ resin. In order to prevent co-elution, we recommend that you perform ion exchange purification prior to the ProBond™ purification. You will need to know the pI of the expressed protein for good binding and need to optimize the ion exchange step for efficient separation from AOX.

We recommend purifying His-tagged Pichia proteins using the protocol described on Pages 50 and 51 in the Pichiamanual , under Purification. It describes how to obtain the supernatant (soluble proteins) and pellet (urea/insoluble proteins) by using the breaking buffer (BB). The composition of the breaking buffer is listed on Page 59 of the Pichiamanual.

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