AC separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a speciﬁc ligand coupled to a chromatography matrix. With such high selectivity and hence high resolution for the protein(s) of interest, puriﬁcation levels in the order of several thousand-fold with high recovery of active material are achievable. Samples are concentrated during binding and the target protein(s) is collected in a puriﬁed, concentrated form. AC can therefore offer immense time-saving over less selective multistep procedures.
In many cases, the high level of purity achieved in AC requires, at most, only a second step on a SEC column to remove unwanted small molecules, such as salts or aggregates.
For an even higher degree of purity, or when there is no suitable ligand for afﬁnity puriﬁcation, an efﬁcient multistep process can be developed using the puriﬁcation strategy of capture, intermediate puriﬁcation, and polishing (CIPP), shown in Figure 9.1.
CIPP is used in both the pharmaceutical industry and in the research laboratory to ensure faster method development, a shorter time to pure product and good economy. AC can be used, in combination with other chromatography techniques, as an effective capture or intermediate step in a CIPP strategy.
This chapter gives a brief overview of the approach recommended for any multistep protein puriﬁcation. The Strategies for Protein Puriﬁcation Handbook, from Cytiva is highly recommended as a guide to planning efﬁcient and effective protein puriﬁcation strategies and for the selection of the correct medium for each step and scale of puriﬁcation.
Figure 9.1. Preparation and CIPP.
Imagine the puriﬁcation has three phases: Capture, Intermediate Puriﬁcation, and Polishing.
Assign a speciﬁc objective to each step within the puriﬁcation process.
The issues associated with a particular puriﬁcation step will depend greatly upon the properties of the starting material. Thus, the objective of a puriﬁcation step will vary according to its position in the process.
In the capture phase, the objectives are to isolate, concentrate, and stabilize the target product. The product should be concentrated and transferred to an environment that will conserve potency/activity.
During the intermediate puriﬁcation phase, the objectives are to remove most of the bulk impurities, such as other proteins and nucleic acids, endotoxins, and viruses.
In the polishing phase, most impurities have already been removed. The objective is to achieve ﬁnal purity by removing any remaining trace impurities or closely related substances.
The optimal selection and combination of puriﬁcation techniques for Capture, Intermediate Puriﬁcation, and Polishing is crucial for an efﬁcient puriﬁcation.
CIPP does not mean that there must always be three puriﬁcation steps. For example, capture and intermediate puriﬁcation might be achievable in a single step, as might intermediate puriﬁcation and polishing. Similarly, purity demands can 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 might 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 of the protein.
Proteins are puriﬁed using puriﬁcation techniques that separate according to differences in speciﬁc properties, as shown in Table 9.1.
There are four important performance parameters to consider when planning each puriﬁcation step: resolution, capacity, speed, and recovery. Optimization of any one of these four parameters can be achieved only at the expense of the others, and each puriﬁcation step will be a compromise (Figure 9.2). The importance of each parameter will vary depending on whether a puriﬁcation step is used for capture, intermediate puriﬁcation, or polishing. Puriﬁcation methods should be selected and optimized to meet the objectives for each puriﬁcation step.
Figure 9.2. Key performance parameters for protein puriﬁcation. Each puriﬁcation step should be optimized for one or two of the parameters.
Capacity, in the simple model shown, refers to the amount of target protein loaded during puriﬁcation. In some cases the amount of sample that can be loaded will be limited by volume (as in SEC) or by large amounts of contaminants rather than the amount of the target protein.
Speed is most important at the beginning of puriﬁcation where contaminant such as proteases must be removed as quickly as possible.
Recovery becomes increasingly important as the puriﬁcation proceeds because of the increased value of the puriﬁed product. Recovery is inﬂuenced by destructive processes in the sample and by unfavourable conditions on the column.
Resolution is achieved by the selectivity of the technique and the efﬁciency and selectivity of the chromatography matrix in producing narrow peaks. In general, resolution is most difﬁcult to achieve in the ﬁnal stages of puriﬁcation when impurities and target protein are likely to have very similar properties.
Select a technique to meet the objectives for the puriﬁcation step.
Choose logical combinations of puriﬁcation techniques based on the main beneﬁts of the technique and the condition of the sample at the beginning or end of each step.
A guide to the suitability of each puriﬁcation technique for the stages in CIPP is shown in Table 9.2.
Minimize sample handling between puriﬁcation steps by combining techniques to avoid the need for sample conditioning. The product should be eluted from the ﬁrst column in conditions suitable for the start conditions of the next column (Table 9.2).
Ammonium sulfate, often used for sample clariﬁcation and concentration (Appendix 1, Characteristics of Ni Sepharose, Ni Sepharose excel, TALON Superflow, and uncharged IMAC Sepharose products), leaves the sample in a high salt environment. Consequently HIC, which requires high salt to enhance binding to the chromatography media, becomes the excellent choice as the capture step. The salt concentration and the total sample volume will be signiﬁcantly reduced after elution from the HIC column. Dilution of the fractionated sample or rapid buffer exchange using a desalting column will prepare it for the next IEX or AC step.
SEC is a nonbinding technique unaffected by buffer conditions, but with limited volume capacity. SEC is well-suited for use after any of the concentrating techniques (IEX, HIC, AC) since the target protein will be eluted in a reduced volume and the components from the buffer will not affect the size exclusion process.
Selection of the ﬁnal strategy will always depend upon speciﬁc sample properties and the required level of puriﬁcation. Logical combinations of techniques are shown in Figure 9.3.
Figure 9.3. Examples of logical combinations of chromatography steps.
For any capture step, select the technique showing the most effective binding to the target protein while binding as few of the contaminants as possible, that is, the technique with the highest selectivity and/or capacity for the target protein.
A sample is puriﬁed using a combination of techniques and alternative selectivities. For example, in an IEX-HIC-SEC strategy, the capture step selects according to differences in charge (IEX), the intermediate puriﬁcation step according to differences in hydrophobicity (HIC), and the ﬁnal polishing step according to differences in size (SEC).
If nothing is known about the target protein, use IEX-HIC-SEC. This combination of techniques can be regarded as a standard protocol.
Consider the use of both AIEX and CIEX to give different selectivities within the same puriﬁcation strategy.