HIC as a Polishing Step

At the polishing stage of a purification protocol most impurities have been removed except for trace amounts or closely related substances such as structural variants of the target protein, nucleic acids, viruses or endotoxins. The purpose of the separation is to reduce these variants and any other trace contaminants to acceptable levels for the application. In contrast to capture steps where a fast, high capacity, step elution is most commonly used, a polishing step will therefore focus on achieving the highest possible resolution.

Media for polishing steps should offer the highest possible resolution. Select as follows:

  1. SOURCE 15 (15 μm mean particle size) — polishing in laboratory or large-scale applications that require high resolution and high throughput (flows up to 1800 cm/h).
  2. Sepharose® High Performance (34 μm mean particle size) if SOURCEmedia do not offer the required selectivity.
  3. Sepharose® Fast Flow (90 μm mean particle size) if the required selectivity is not available in a medium of smaller particle size.

Optimize the gradient elution to maximize selectivity. Use high-efficiency media with small bead sizes to improve resolution.

Note that if HIC is used as a polishing step, it may be necessary to remove excess salt using a desalting/buffer exchange step.

Purification of a recombinant Pseudomonas aeruginosa exotoxin A, PE553D

Figure 56 shows a four-step purification process, using expanded bed adsorption (EBA) followed by HIC-IEX-HIC, for the purification of a genetically modified recombinant P. aeruginosa exotoxin A (Mr 66 000) expressed in the periplasm of E. coli. The strategy used here resulted in a highly purified exotoxin A that took less than half the time of a conventional approach.

Four-step purification process

Fig 56. Four-step purification process.

Exotoxin A was captured directly from unclarified E. coli homogenate by expanded bed adsorption using STREAMLINE DEAE adsorbent in STREAMLINE 200 column. The collected fraction was transferred directly to an intermediate purification step using step elution on Phenyl Sepharose® 6 Fast Flow (high sub) to remove a substantial part of the UV absorbing material that could interfere with the following steps (Figure 57a). Note that a Sepharose® 6 Fast Flow matrix was used, rather than Sepharose® High Performance, as this was a large-scale purification and a rapid step elution rather than a higher resolution gradient elution was required.

A second intermediate purification used an anion exchanger, SOURCE URCE 30Q, to remove the majority of the remaining contaminants, based on differences in their net surface charge (Figure 57b).

HIC was used again for the polishing step, this time using a gradient elution to take full advantage of the high resolution offered by the smaller particle size of SOURCE 15PHE (Figure 57c). The process resulted in a pure protein, according to PAGE and RPC analysis, and the overall recovery was 51%.

Purification of Exotoxin A

Fig 57. Purification of Exotoxin A.

Alternative techniques for polishing steps

Most commonly, separations by charge, hydrophobicity or affinity will have been used in earlier stages of a purification strategy so that high-resolution gel filtration is ideal for the final polishing step. The product can be purified and transferred into the required buffer in one step, and dimers and aggregates can be removed, as shown in Figure 58.

Gel filtration is also the slowest of the chromatography techniques and the size of the column determines the volume of sample that can be applied. It is therefore most logical to use gel filtration after techniques that reduce sample volume so that smaller columns can be used. Media for polishing steps should offer the highest possible resolution. Superdex® is the first choice or gel filtration at laboratory scale and Superdex® prep grade for large-scale applications.

Fig 58. Final polishing step: separation of monomers, dimers and multimers on Superdex 75 prep grade.

RPC can also be considered for a polishing step, provided that the target protein can withstand the run conditions. Reversed phase chromatography (RPC) separates proteins and peptides on the basis of hydrophobicity. RPC is a high-selectivity (high-resolution) technique, usually requiring the use of organic solvents. The technique is widely used for purity check analyses when recovery of activity and tertiary structure are not essential. Since many proteins are denatured by organic solvents, RPC is not generally recommended for protein purification because recovery of activity and return to a correct tertiary structure may be compromised. However, in the polishing phase, when the majority of protein impurities have been removed, RPC can be an excellent technique, particularly for small target proteins that are not often denatured by organic solvents.

Cipp does not mean that there must always be three purification steps. For example, capture and intermediate purification may be achievable in a single step, as may intermediate purification and polishing. Similarly, purity demands may be so low that a rapid capture step is sufficient to achieve the desired result. For purification of therapeutic proteins, a fourth or fifth purification step may be required to fulfill the highest purity and safety demands. The number of steps used will always depend upon the purity requirements and intended use for the protein.