7.1.1 The Theory of Chromatographic Separation

The mobile phase flow and analyte separation when applied to chromatography can be explained by several main mathematical relationships and physical parameters. Essentially a chromatogram, the signal response product of a chromatographic separation, is a plot of analyte concentration versus eluent volume or, less fundamentally, time (Figure 7.2). The retention time (t _r ) or volume for a particular analyte is an important parameter for characterising the separation. Retention time is dependent on the volumetric flowrate. However, the retention time also depends on the dimensions of the column used. Therefore, the retention time is usually reported as a normalised retention quantity known as the capacity factor (or retention factor) (k). Selectivity (or separation factor) (α) is the ratio of the retention (capacity) factors (k) of two peaks. The efficiency (N) is reported as the number of theoretical plates for a column. Traditionally, a ‘plate’ (the name is taken from the ‘plates’ used in fractional distillation towers used in the petroleum industry) is defined as a hypothetical separation layer within the column in which the two phases establish equilibrium with each other. The higher the number of plates the more efficient the separation will be. The concept of a layer in which equilibrium is established is a rather abstract one in that chromatography is a dynamic process and equilibrium is never reached. For simplicity it may be useful to note that analytes elute from the chromatography column as a peak with a Gaussian distribution and the plate height is related to the variance of that peak. The Gaussian distribution arises from the band broadening processes that take place in the column, giving rise to random error in the time taken for molecules of a single analyte to elute from the column. Resolution (R) is defined as the separation of two analyte peaks, taking into account the individual peak widths. The main mathematical relationships used in chromatographic separation are shown below:

where

• k is the retention factor
• t _r is the retention time for a particular analyte (e.g. r = 1 for the first peak)
• t ₀ is the column void volume (solvent front)
• N is the number of theoretical plates
• w _n is the width of an individual peak
• R _s is the resolution of two individual peaks

The above equation for resolution is used for the actual measurement of resolution from a chromatogram. This should not be confused with an alternative equation for resolution, which may be obtained by substituting the other three equations into the equation for resolution.

The Purnell equation [31] describes the resolution of two similar peaks under chromatographic separation taking into account the selectivity, capacity and plate number:

The Purnell equation is an extremely useful relationship that can be used to gain a deeper insight into what qualitatively is obvious. Better resolution may be had by having narrower peaks (better efficiency) and by having a larger ratio of capacity factors (selectivity). As the Purnell equation also indicates, better resolution may be had by increasing the capacity factor. However, not only is this at the expense of increasing analysis time but there is little benefit in terms of increasing resolution for k > 10.

The separation of two analytes (Figure 7.2) is only possible if their retention times differ by virtue of having different distribution coefficients between the mobile phase and stationary phase. While the principal goal in this case is to obtain reliable baseline resolution in as short a time as possible, an associated goal is to obtain this not just through good peak efficiency but also through good peak symmetry; i.e. there may be deviations from the anticipated symmetrical Gaussian peak shape in the form of fronting and/or tailing. There are several main causes for non‐Gaussian peak shapes, which include: column stationary phase deterioration, column under‐ or overpacking, mobile phase impurities or loss of liquid handling control and/or efficiency [32,33]. Peak width is usually measured at both the baseline inflection points and at half of the maximum signal height (Figure 7.2). By monitoring peak width over repeated separations, both column and system performance may be determined. For example, if a column is still being conditioned with the sample there will be a reduction in peak width over time. Also of note is the fact that as the concentration of an analyte in the sample increases there is usually a consequent broadening of the peak (mass overloading). Other factors leading to an increased peak width are dilute sample injections over long periods of time (usual in preparative protein chromatography and volume overloading), very shallow or elongated gradient elution profiles, mobile phase impurities or incorrect make‐up and/or problems with liquid handling.

Gradient elution is in contrast to isocratic elution. When eluting analytes in chromatographic separations, there are three distinct types of elution (Figure 7.3) available. Isocratic elution utilises one mobile phase consistently across the whole separation. Isocratic elution is used in size exclusion chromatography but is rarely used when other modes of liquid chromatography that are used for the separation of large biologicals. This applies in particular to preparative liquid chromatography. Gradient elution utilises two (or very occasionally more) mobile phases and over a period of time gradually mixes the phases so that the composition changes from the initial composition to a new composition. For the majority of liquid separations, the gradient is linear. Step elution, like gradient elution, utilises two (or more) mobile phases, but rather than gradually changing the composition it uses a series of ‘step‐up’ transitions to achieve a new mobile phase composition. In the analysis of small molecules a gradient is commonly used to shorten run times by bringing strongly retained sample constituents off the column more quickly. In preparative work on large biologicals the application of a gradient is more commonly used to facilitate separation and aid resolution of closely related analytes. Step elution is used to generate fast but crude ‘cuts’ of adsorbed species from the column. It is common to use step elution after the mobile phase composition needed for a specific analyte elution has been characterised via gradient elution.

In considering the theory of chromatography in the context of biological applications it must be remembered that it is not just about manipulating retention, selectivity and efficiency to obtain resolution of large molecules. Biological samples, particularly those containing small molecules, can be very complex, say several hundreds of thousands of components compared to maybe tens of components in a small molecule drug sample. A real problem can be that, even with very narrow peaks, there are simply too many sample constituents for them all to be resolved in a given run time. In other words, there is insufficient peak capacity. Temperature, flow rate, use of smaller particles, etc., can be used to increase peak capacity. However, for a step improvement it is necessary to use multidimensional chromatography where the peak capacity is the product of the peak capacity of each dimension. For this reason, comprehensive 2D‐LC (very small volumes of eluent from the first column are all chromatographed on a second column with different selectivity in real time) is attracting great interest [34]. In the meantime, in the quantitative determination of complex biological samples it is almost the norm to add another dimension when using a single column through the use of a mass spectrometer as the detector. Mass spectrometry in its own right is a powerful tool in the analysis of biologicals (Chapter ) but while the focus here is more on the actual chromatography of biological molecules, including for preparative purposes, it cannot be emphasised enough that liquid chromatography in combination with mass spectrometric detection is very powerful for the characterisation and quantitative determination of biologicals.

7.1.2 Chromatography for Biological Samples

Biological samples are, by their very nature, complex, containing impurities that are analyte‐related (minority) and analyte‐unrelated (majority). Analytical and preparative separation methods need to be robust in terms of their reproducibility and, for preparative work, throughput (multiple injections/loadings), while maintaining a selectivity for a physicochemical property for the analyte. Chromatography as a separation technique can provide this selectivity requirement even when applied to heterogeneous sample mixtures from a variety of sources.

Chromatography can also be used in‐line with other preparative and analytical techniques since it can provide the analyte in an eluent that is suitable for further downstream separation and analysis (for example spectrophotometry, electrophoresis, circular dichroism, nuclear magnetic resonance [NMR] and mass spectrometry).

However, the dynamic nature of biological samples can be a technical challenge when applying any form of separation technique. Ensuring that the sample applied to the chromatographic technique is indeed representative of the true biological system from which the sample was taken needs to be considered at all stages of sample preparation through to data analysis. When applied correctly, chromatography can be non‐destructive, modulated in terms of physical properties (pH, temperature, conductivity), have efficient sample processing times, and take advantage of computation integration and processing methods to circumvent many of these challenges.

7.1.3 Biological Sample Preparation for Chromatography

While there are chromatographic applications for isolating [35] and characterising intact cells [36], in just about all cases for chromatographic separation of biological samples some initial form of cellular disruption is required [37]. Disruption methods will release various compartmentalised hydrolases (phosphatases, glycosidases and proteases) that can alter the composition of the lysate [38]. In all modern experimental approaches, it is essential to preserve sample composition from the point of generation to analysis. Indeed, managing sample complexity can be an added challenge when working with biological material [39,40]. Even the most sophisticated modern instrument cannot produce meaningful data without some form of rational sample preparation. Biological samples, irrespective of their origin, will contain a heterogeneous mixture of small and macromolecular species (Table 7.2), all of which will behave differently when applied to chromatographic separation.

At the start of the sample preparation phase, there are several important considerations that need to be taken into account since they impact the sample preparation strategy:

• Measurement: The application of a simple measurement like UV‐spectrophotometry may still incur problems if the sample does not have a chromophore or if the chromophore is masked by sample impurities leading to coelution and/or signal broadening. The application of more sophisticated measurement techniques like mass spectrometry [5] or NMR spectroscopy [50] may require the removal of specific sample constituents prior to their use, not to mention other associated technical considerations (Table 7.3).
• Quantitation: Accurate quantitation is needed for all chromatographic separations. The level of quantitation can range from basic peak area determination to more complex comparisons with internal standards to generate accurate titre values for the analyte and in some cases the main impurity peaks.
• What is the sample composition and how much is known about it? The organic versus inorganic, solid versus soluble sample composition is by far the most important property of the sample to consider prior to chromatographic separation. Of course, it goes without saying that if the sample is solid or a mixture of solid and liquid then exactly how subsampling is carried out is a major consideration. Failure to account for sample composition can lead to poor chromatographic separation and even failure of the chromatographic media and liquid pumping systems. The sample volume and viscosity will affect the solubility of many analytes and this may change as a sample is stored based on the environmental conditions.

Only after addressing these considerations can an appropriate strategy for sample pre‐treatment be determined.

7.1.4 Extraction and Cleanup Methods for Chromatographic Sample Preparation

Through sample extraction, it is possible to change the sample composition to suit the chromatographic separation method and/or reduce the sample complexity quite significantly. Common sample extraction methods used for biological samples prior to chromatographic separation (Table 7.4) and subsequent sample cleanup techniques that can be applied to an extracted sample (Table 7.5) can, in many cases, be applied in series to yield a sample that is ‘clean enough’ to be further processed via chromatographic separation.

7.1.5 Protein Separation

As alluded to earlier, applications of chromatography to ‘biology’ are many and varied. However, the analysis of proteins has always been of paramount importance. Biochemists have been studying proteins since the early nineteenth century and the efficient chromatographic separation techniques in use today were developed in the early 1940s and 1950s and porous organic polymers were developed as part of the Manhattan Project (to develop the first atomic bomb!). Tiselius developed the basic chromatographic principles of chromatographic frontal analysis (continuous feed of the sample solution; the first constituent that breaks through may be obtained pure) in the early 1940s and applied a refined set of these techniques to the analysis of ovalbumin, serum albumin and immunoglobulins in the 1950s. In the subsequent six decades many different forms of chromatographic separation for protein preparation (purification) and analysis (concentration and structural determination) have been developed.

7.2.1 Size/Shape – Exploited by Size Exclusion Chromatography (SEC)

In size exclusion chromatography (SEC) (Figure 7.4a and b) the molecular sieving properties of porous materials are used to separate proteins based upon their size and shape. Traditionally in SEC, a column of microparticulate cross‐linked copolymers (styrene or divinylbenzene) and with a narrow range of pore sizes is in equilibrium with the mobile phase. More recently, it is common to use silica‐based particles typically with an immobilised diol surface for protein SEC. Large analytes are excluded from the pores and pass through the spaces between the particles and are the first analytes to elute from the column. Smaller analytes are distributed between the mobile phase inside and outside the particles and will therefore take longer to pass through the column. The smallest analytes are the last to elute from the column. The mobile phase within a particle is available to an analyte that is able to penetrate the particle. The distribution of an analyte in a SEC column is determined by the total volume of mobile phase inside and outside the particles. For a given type of particle, the distribution coefficient (K _d ) of a particular analyte between the inner and outer mobile phase is a function of its molecular size. If the analyte is large and completely excluded from the mobile phase within the particle, K _d = 0. If the analyte is sufficiently small to gain complete access to the inner mobile phase, K _d = 1. However, a variation in pore size will mean that the K _d values vary between 0 and 1. It is this complete variation of K _d between these two limits that makes it possible to separate analytes within a narrow molecular size range on a given particle type. It should be noted that since the protein separations take place before t ₀, SEC columns are usually longer and wider than those for other modes of chromatography.

The sizing properties of SEC make it a very powerful technique for characterising proteins in their native conformation, but SEC can also be applied for desalting protein preparations and concentration of particular analytes. The application of SEC has also been successful for separating nucleic acids [ 6,87], viral particles [88], large molecular weight formulation products [89] and multicomponent cellular assemblies [90]. For all of the applications, good knowledge of common SEC stationary phase polymers and the associated technical considerations (Table 7.6) is important.

Mode	Stationary phase description and technical considerations
Size exclusion chromatography (SEC)	Low pressure stationary phase polymers: Dextran (Sephadex, Sephacryl), Agarose (Sepharose) and Polyacrylamide (Bio‐gels) High pressure stationary phase polymers: Polyvinyl chloride (Fractogel) and Dextran (Superdex) SEC resins are prone to compression and sometimes difficult to pack. For some applications, fine resolution is only obtained with excessively long columns (30–100 cm), which can be prohibitive on many systems. SEC columns should be cleaned and stored in 200 mM sodium acetate, 20% v/v ethanol. 0.5 M NaOH can be used for brief robust cleaning if needed.
Ion exchange chromatography (IEX)	Anion exchangers: Strong: Trimethylaminomethyl: ‐O‐CH2N+(CH3)3 (cellulose matrix) Triethylaminoethyl: ‐O‐CH2CH2N+(CH2CH3)3 (dextran or cellulose matrix) Weak: Aminoethyl: ‐O‐CH2CH2N+H3 on an agarose matrix Diethylaminoethyl: ‐O‐CH2CH2N+(CH2CH3)2H (cellulose matrix) Cation exchangers: Strong: Sulpho: −SO3 −, O‐CH2SO3 −, O‐CH2CH2CH2SO3 − (cellulose, dextran, or polystyrene matrix) Weak: Carboxymethyl: O‐CH2COO− (cellulose or dextran matrix) It is important that IEX columns are adequately equilibrated prior to sample application so as to bind and prepare all available functional groups with a counter ion (for proteins this would be Na+Cl‐) and to apply a set mobile phase pH across the entire column. IEX can be used in gradient or step elution modes and elution can be via pH changes or counterion concentration changes. A final high ionic strength wash after the elution step will allow the column to be cleaned and regenerated for subsequent reuse. 0.5 M NaOH can be used for brief robust cleaning. IEX columns should be stored in 20% v/v ethanol between uses.
Hydrophobic interaction chromatography (HIC)	Common HIC and RPC stationary phase chemistries: The performance of HIC separation is mostly dependent on the properties of the protein and the stationary phase chemistry. The stronger the ligand hydrophobicity and/or the more surface hydrophobicity on the protein the stronger the interaction. However, these interactions are also affected by the presence of contaminating analytes, detergents, pH and the ligand density/degree of substitution. The dynamic binding of the analyte and the subsequent resolution is determined by the flowrate, salt concentration and mobile phase properties. Complete elution of bound species can be a problem if a very hydrophobic ligand is matched with a high salt concentration in the injection sample. Selecting a less hydrophobic ligand or a less substituted matrix can help solve this problem. Temperature can have an effect on some complex sample separations also. Columns must not be left with bound analytes and can be fully eluted and cleaned by 1 M NaOH and are usually stored in 20% v/v ethanol between uses.
Affinity chromatography	Established affinity chromatography applications: Purification of kinase and dehydrogenase enzymes through the immobilisation of nucleotides (5′‐AMP and 2′5′‐ADP) Purification of heparin binding proteins (growth factors, DNA, lipoproteins, membrane receptors) via the immobilisation of heparin Isolation and identification of DNA binding proteins through the immobilisation of DNA (specifically promoter sequences and regulatory elements) Isolation of fatty acid binding proteins through the immobilisation of fatty acids Isolation of immunoglobulins through the immobilisation of Protein A and Protein G ligands Isolation of mRNA through the immobilisation of poly‐Thymine Isolation of recombinant proteins through the engineering of fusion protein partners and metal affinity binding motifs Elution of the bound protein (or other analyte) needs to be complete to ensure performance upon reuse. Elution is usually through the application of high ionic strength buffers (1.5 M KCl or 2 M NaCl) or changes to the pH of the mobile phase that change the surface charge of the protein and allow it to dissociate from the column. Cleaning of the resin can be carried out using detergents, chaotropes in low or high pH buffers containing 1.5 M NaCl. Strong acids and bases should not be applied to affinity resins so as to maintain the ligand integrity. Storage should be in 20% v/v ethanol.

7.2.2 Charge – Exploited by Ion Exchange Chromatography (IEX)

This form of chromatographic separation relies on charge–charge interactions between the analyte and the stationary phase. Ion exchange chromatography (IEX) (Figure 7.5a and b) is a very versatile form of chromatography that can be applied over a wide range of biomolecules, synthetic polymers and small molecules. Both cation and anion exchanger stationary phases have a high binding capacity and their retention and resolution can be modulated through the ionic strength and pH of the mobile phase. The exchangers are either acidic (cation exchanger, negatively charged) or basic (anion exchanger, positively charged). Analytes are exchanged on the stationary phase through ionic interactions (a positively charged analyte interacting with a negatively charged stationary phase functional group, and vice versa). IEX can be carried out using either a strong or weak exchanger, wherein the strength of the exchanger refers to the extent to which the ionisation state of the functional groups varies with pH. It is not the strength of the interaction between the stationary phase and the analyte. The selection of either a weak or strong exchanger (Table 7.6) depends on the nature of the separation. If the separation is using a wide pH range (gradient pH‐based elution) then a strong exchanger is preferable as it ensures that retention via ion exchange will take place at both extremes of pH. Weak anion exchangers should be used when the binding capacity of the stationary phase is known to change as the pH of the mobile phase varies. In most IEX method development exercises it is advisable to select and screen strong exchangers first before moving to a weak exchanger to, if required, introduce an additional element of selectivity.

For protein separation via IEX the isoelectric point (pI, wherein the protein has no overall net charge) of the protein of interest should be obtained as this will allow an appropriate exchanger and mobile phase to be selected. This is particularly important if a pH gradient is being used instead of a buffer gradient. At a pH lower than the pI, the protein will have a net positive charge (suitable for cation exchange), while at a pH above the pI the protein will have a net negative charge (suitable for anion exchange). However, the stability of the protein as pH varies and the physicochemical properties of any putative impurities in the sample should also be considered so as to ensure that chromatograms obtained are representative of the initial sample. IEX may also be successfully employed for the separation of nucleic acids [91], drugs and impurities [10].

7.2.3 Hydrophobicity – Exploited by Hydrophobic Interaction Chromatography (HIC) and Reversed‐Phase Chromatography (RPC)

Hydrophobic interaction chromatography (HIC) (Figure 7.6a and b) has been applied for protein separation for over 30 years [92,93]. Separation is based on the adsorption of hydrophobic areas on the surface of an analyte on to the stationary phase (or, in some models, partition into a less polar area of mobile phase close to the stationary phase). For proteins the majority of hydrophobic residues are buried in the centre of the protein protected from the aqueous environment of the surface; indeed, it is the formation of this hydrophobic core that drives protein folding in globular proteins. Hydrophilic amino acids on the protein surface are covered with an ordered layer of water. Hydrophobic ‘patches’ on a protein surface can be exposed by treating the protein with salt ions (usually ammonium sulphate), which preferentially take up the ordered water molecules and allow the hydrophobic areas to be exploited for capture and separation activities. Interactions are via weak van der Waals forces between the protein and the stationary phase. However, protein–protein interactions are possible via the exposed hydrophobic areas and may cause aggregation of the protein and eventual precipitation. HIC can be applied in both low pressure protein capture (selective initial isolation step) and high pressure analysis formats. For low pressure chromatography, HIC makes an ideal intermediate or polishing step (further treatment of the ‘captured’ protein) in protein purification. The binding of proteins to an HIC stationary phase is normally in high salt concentrations and elution proceeds via the gradual decrease in salt concentration. This gradual change in salt concentration will cause a rehydration of the protein surface and a loss of interaction with the stationary phase. Changing the mobile phase pH can also be used to elute HIC separations, but the resulting separation can be quite variable for complex samples as this changes the separation to a more mixed mode (HIC:IEX) format and requires specialised stationary phase chemistries, which must have good batch‐to‐batch reproducibility. When applied in an HPLC format, HIC can be used to separate very closely related analytes using very shallow gradients and small resin beads or particle sizes (≤5 μm). The stationary phases commonly used for HIC are naturally hydrophobic, containing groups such as butyl, octyl and phenyl attached to a matrix, which promotes more protein–matrix interaction rather than protein–protein interactions. This quality of HIC makes it very versatile and able to accommodate high loading capacities. Common stationary phases include Phenyl Sepharose and Phenyl‐5PW for low pressure HIC and Bio‐Gel TSK Phenyl and Spherogel TSK Phenyl for HIC HPLC.

HIC is very similar to reversed‐phase chromatography (RPC, also commonly abbreviated as RPLC or RP‐HPLC). However, in RPC the mobile phase will contain a polar‐organic constituent, such as methanol or acetonitrile, that may promote sample denaturation, especially for proteins. RPC tends not to be suitable for protein purification, except in very special circumstances [79], but is a very versatile and commonly used application for protein analysis. RPC has a lower binding capacity than HIC. While HIC is commonly used for protein separation it has also been applied to lipids [94], carbohydrates [95] and nucleic acids [96]. Common HIC and RPC stationary phase chemistries (Table 7.6) can be used in either HIC or RPC but generally more hydrophobic chemistries are used in RPC with polar organic solvents in the mobile phase and less hydrophobic chemistries are used in HIC with highly or completely aqueous buffer containing mobile phases.

RPC is very much the most common mode of chromatography for small molecules [97]. Therefore, as far as proteins are concerned, RPC comes into its own in the elucidation of protein structure where it is used for peptide analysis of protein tryptic digests (Section 7.4).

7.2.4 Biological Function and/or Structure – Exploited by Affinity Chromatography

Affinity chromatography (Figure a and b) exploits the biological function or specific structure of an analyte for separation making it extremely specific and capable of purification to high purity from very complex sample mixtures [17]. It is this specificity that makes affinity chromatography very expensive to use. Affinity chromatography is probably best characterised by the purification of enzymes by immobilising substrates/ligands (Table 7.6) on to chromatography stationary phase matrices. The enzymes would bind to the immobilised substrate once introduced to the column [98,99]. The interaction between the stationary phase ligand and the analyte needs to be reversible so that the column can be reused and so the analytes can be eluted. Elution is usually carried out through the application of free‐ligand changes in pH or salt gradients. Affinity chromatography is now used as the primary capture step for purifying immunoglobulin molecules used for therapeutic applications, but is also used for separating nucleic acids, membrane‐bound proteins and even whole cells. The use of affinity methods requires a detailed knowledge of the biological function and to some extent structure of the analyte of interest. The stationary phases need to be relatively inert in terms of their binding characteristics but possess suitable chemistry to allow attachment of the ligand. In this way retention is exclusively by the affinity interaction and resolution is not lost under the influence of non‐specific interactions. Ligands are usually added along with a spacer of up to 10 units (usually hydrocarbon chains but can contain carbonyl or amine groups) in length to allow correct orientation of the ligand and stop steric hindrance during binding to closely immobilised ligands. The matrix is usually an agarose or dextran base. Unlike other forms of chromatographic separation, affinity chromatography can be carried out in both batch‐resin and packed‐column formats.

7.4.1 On‐Column Protein Folding

• Rationale: As described earlier (Section 7.3), the rapid expression of proteins in E.coli cell cultures can lead to the formation of inclusion bodies. Historically, these protein aggregates were seen as ‘lost’ products and efforts were made to try to express the protein in a more soluble format. However, the relatively high purity obtained through the generation and isolation of inclusion bodies has more recently made them more attractive for protein purification. There are many solubilisation and refolding protocols available for application to many proteins expressed as inclusions bodies. However, for some proteins the solubilisation and refolding procedures lead to a loss in yield and/or a reduction in product quality. On‐column refolding can in many circumstances circumvent these problems through stabilisation of the protein during the molecular shaping into its final conformation. In addition, on‐column refolding allows processing at much smaller volumes (refolding is usually carried out in volumes that are 20‐fold larger than the solubilisation and therefore necessitate large column loadings of dilute protein samples) and aids in the analysis and preparation of proteins generated via high throughput screening (HTS) technologies [116]. During method development activities combining the refolding and primary capture chromatography steps can simplify an overall purifications scheme. However, the on‐column refolding step has been employed as part of the primary capture chromatography step and after an initial chromatographic capture and crude purification.
• Technical considerations: The application of on‐column protein refolding is dependent on the protein of interest, the available time, money and chromatographic hardware available. For inexperienced users it may be prudent to first define a test‐tube refolding protocol and then capture the column step for a refolding product and attempt to combine the two steps. Due to its high capacity and robust stationary phase chemistry, IEX is an ideal capture and separation mode for refold samples. The stationary phase will be exposed to chaotropes (e.g. 8 M urea or 5 M guanadine‐HCl), reducing agents (e.g. 10 mM dithiothreitol [DTT]), oxidising agents (e.g. 5 mM cystamine‐HCl) as well as extremes of pH, and as such will need to be properly cleaned, regenerated and stored after use. Both strong and weak exchangers can be applied and it is common to screen several exchangers for optimisation. Proteins should be loaded on to the column at concentrations of ≤10 mg/ml. The solubilisation sample should be carried out with ‘clean’ inclusion bodies that are devoid of extraction buffer components, e.g. detergents. Protein folding can be a spontaneous process both in vivo and in vitro in test‐tube preparations, but for immobilised protein it is advisable to slowly introduce the refolding environment through the application of a gradient. Elution is carried out using high salt buffers (0.5–1 M NaCl) or a change in the mobile phase pH.
• Simplified method: A baseline AIEX protocol for on‐column refolding.

7.4.2 On‐Column Protein Refolding via Anion Exchange Chromatography

1. Wash the inclusion body preparation to achieve a clean ‘white’ protein mass – this can be done with water or aqueous buffer/1 M urea mix.
2. Store inclusion bodies as a 25–50% w/v slurry in the washing solution. This slurry can be stored at −80 °C prior to chromatographic processing.
3. Prepare the solubilisation buffer (50 mM Tris‐HCl, 8 M urea, 10 mM DTT, pH 9.0) and then slowly add the inclusion body slurry to a final concentration of 10 mg/ml protein. Allow the solubilisation to mix for 30–60 minutes. Note that if a cation exchange chromatography (CIEX) column is used then the base buffer should be modified (e.g. sodium acetate).
4. Equilibrate an anion exchange column with five column volumes of solubilisation buffer and then load the solubilisation mixture on to the column. Wash the column with five column volumes of solubilisation buffer.
5. Refold the bound protein by applying the refold buffer (50 mM Tris‐HCl, pH 9.0) from 0 to 100% over 10 column volumes.
6. Elute the column using a step elution with 50 mM Tris‐HCl, 0.5 M NaCl, pH 9.0.
7. The column should be cleaned and stored after use.

For an example on‐column refolding of recombinant lysozyme (Figure 7.8a), showing the associated size exclusion separation of key elution samples from the on‐column refolding (Figure 7.8a), the diethylaminoethanol (DEAE) elution of the refolded lysozyme is resolved into conjoined two peaks. The peak 1 refolded sample shows a shift in retention time (16 minutes) compared to the solubilised load material (main peak max. at 7 minutes). The solubilisation sample resolves into two conjoined peaks (peak max. at 7 and 10 minutes, respectively). The DEAE elution peak 2 contains higher molecular weight material (lower retention time) with a similar retention time peak max. of 10 minutes to some of the solubilised material. The DEAE column cleaning step contains high molecular weight material similar to the solubilisation sample (peak max. at 7 minutes). The conclusions from this on‐column refolding experiment are that lysozyme can be captured post‐solubilisation and refolded using on‐column methods. The post peak material seen on the DEAE column contains higher molecular weight material that could be aggregates or misfolded protein. The cleaning step removes material that is likely to be non‐refolded protein.

Associated analytical technologies: Assaying for protein structure can be a relatively complex undertaking. However, there are some techniques (circular dichroism, protein NMR or hydrogen‐deuterium exchange mass‐spectroscopy) that will provide detailed information on the folding dynamics and crucially the final 3D conformation of the protein. However, there are simpler analytical technologies that can be employed to monitor the changes in protein conformation, for example analytical ultracentrifugation and size exclusion chromatography.

7.4.3 Protein Structure Elucidation

Even after isolating an intact protein, it may still be difficult to characterise the purified product. However, such a ‘top‐down’ study of a large molecule can be complemented by a ‘bottom‐up’ approach to protein analysis. This could involve, for example, papain digestion of a monoclonal antibody, but this results in a few still relatively large fragments. On the other hand, deglycosylation of monoclonal antibodies allows glycan analysis by hydrophilic interaction liquid chromatography (HILIC) liquid chromatography–mass spectrometry (LC‐MS) to study the sugars responsible for molecular recognition, HILIC being a relatively recently popularised mode of LC involving a polar stationary phase and a mobile phase containing a high proportion of a polar organic solvent and a low proportion of aqueous buffer that is suitable for polar analytes and is characterised by good MS sensitivity [117]. This type of analysis is very valuable as it is post‐translational modifications such as glycosylation that endow proteins with many of their important properties. This is particularly the case in the study of monoclonal antibodies and ‘biosimilars’ used as biopharmaceutical drugs.

However, the most common form of the ‘bottom‐up’ approach is to carry out a tryptic digest of the protein and subsequently characterise the product peptides by LC (Figure 7.9) or, more frequently, LC‐MS or even 2D‐LC‐MS. This chromatography approach is now widely used and the chromatography used has been perfected to the point that the actual digestion is a significant variable. To this end, manufacturers are now introducing kits to carry out solid phase digestion to reduce the variability caused by time, temperature and autocatalytic digestion of the enzyme.

An even greater challenge is to determine low levels of biopharmaceutical drugs in biological fluids. This can be and is carried out on the intact protein but even here the bottom‐up approach can be applied. A signature product peptide may be found for a particular biopharmaceutical and levels of that signature peptide may be monitored as a surrogate for the biopharmaceutical.

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Further Reading

1. Conductivity E, Tds S (2004) 760. Electrical Conductivity/Salinity Fact Sheet. Water Resour 0:2–6.
2. Dong, M.W. (2006). Modern HPLC for Practising Scientists. Wiley.
3. Gooding, K.M. and Regnier, F.E. (2002). HPLC of Biological Macro‐ Molecules, Revised and Expanded. CRC Press; Taylor and Francis Group.
4. Grigore, M.‐N., Ivanescu, L., and Toma, C. (2014). Halophytes: An Integrative Anatomical Study. Springer International Publishing.
5. Snyder, L.R. (1992). Chapter 1, Theory of chromatography. In: Journal of the Chromatography Library, A1–A68.
6. Snyder, L.R., Joseph, J.K., and Dolan, J.W. (2010). Ionic samples: reversed‐phase, ion‐pair, and ion‐exchange chromatography. In: Introduction to Modern Liquid Chromatography, 303–360. Hoboken, NJ, USA: Wiley.
7. Striegel, A.M., Yau, W.W., Kirkland, J.J., and Bly, D.D. (2009). Modern Size Exclusion Chromatography: Practice of Gel Permeation and Gel Filtration Chromatography, 2e. Wiley.
8. Timms, J.F. and Cutillas, P.R. (2010). Overview of quantitative LC–MS techniques for proteomics and activitomics. Methods Mol. Biol. 658: 19–45.
9. Walls, D. and Loughran, S. (2011). Protein Chromatography: Methods and Protocols. Springer.