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Ion Exchange with PureSpeed TipsA Powerful Chromatography Tool

Ion exchange chromatography separates molecules by exploiting differ-ences in their overall charge characteristics. Its simplicity makes this type of chromatography a powerful tool for separating proteins, nucleic acids, and other molecules such as peptides. With no special tagging of biomol-ecules needed, ion exchange is a good general solution for purification. The buffer conditions used in ion exchange are also usually compatible with the molecules of interest, so functional activity is preserved during the purification process.

The PureSpeed purification system includes a Rainin E4 XLS pipette and pipette tips containing resin capable of carrying out ion exchange chroma-tography. When set up with a deepwell plate containing appropriate buffers and samples, the PureSpeed system is able to carry out standalone up-and-down pipetting that drives the movement of purification solutions over one of four ion exchange resins. When a given step is complete, the user simply moves the pipette to the next row of wells in the deepwell plate. PureSpeed for ion exchange system offers many benefits, including:

• Fast purification – depending on user needs, purifies in as little as 10 minutes.

• Many purification options – strong and weak anion and cation exchange.

• Multivariable purification screening – perform strong and weak anion and cation exchange at the same time, while scanning purification conditions such as salt and pH.

• Parallel processing – unlike FPLC PureSpeed can process up to 12 samples at the same time.

• More convenient than gravity and spin columns – semi-automation eliminates the need for constant attention and a centrifuge.

• Small bed volumes – higher concentration final sample due to Pure-Speed's small elution volume.

• Flexible – can also be used for DNA oligonucleotides, peptides, and small molecules.

• Quickly purify or polish multiple samples – polishing with FPLC is time-consuming for multiple samples.

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eIon Exchange Chromatography Principles

Four types of ion exchange resins are commercially available: strong anion, strong cation, weak anion and weak cation exchange resins (Figure 1). These resins are differentiated by the func-tional groups that are appended on the surface of the individual resin beads. Strong and weak anion exchange resins have either quaternary amines or tertiary amines attached to their sur-faces respectively. These groups are positively charged and for weak ion exchange resins, the charge of the tertiary amine can be removed by increasing the pH of the mobile chromatogra-phy phase. Strong and weak cation exchange resins either have sulfonic or carboxylic acids attached to their surfaces, respectively. For cation exchange, the immobilized groups are neg-atively charged and for weak cation exchange the charge of the carboxylic acid can be masked by decreasing the pH.

Figure 1. Ion Exchange Resin Types Anion exchange resins are positively charged, thus can form ion pairs with anionic biomolecules. Removal of anionic biomolecules from the positively charged resin is performed with a competing anion, such as chloride anion from sodium chloride solution. At high concentrations, chloride competes for the anion exchange resin and the anionic biomolecule is released. Though the pKas of the ionizable groups are provided, the working pH for anion exchange resins is either 2 to 9 for weak anion exchange, or 2 to 12 for strong anion exchange. Conversely, cation exchange resins display negatively charged molecules so that ion pairs can be form with positively charged biomoecules. Displacement of positively charged biomolecules from the negatively charged resin is per-formed with a competing cation, such as sodium ion from sodium chloride solution. At high sufficiently high concentrations, so-dium displaces the cationic biomolecule from the cation exchange resin and the biomolecule is eluted. The pKas for each resin are provided, but the working pH for cation exchange resins are 6 to 10 for weak cation exchange, and 4 to 13 for strong cation exchange.

Ion exchange chromatography relies on the interaction of charged biomolecules with oppositely charged surfaces on purification resins. For a protein or oligonucleotide, exposed surface charges on each type of molecule partly dictate the binding affinity towards a given resin (Table 1). The net surface charges of proteins and other biomolecules depends on the chemical com-position of the species. Proteins, for instance, are composed of amino acids that assume dif-ferent charges depending on the solution pH. At solution pH values greater than the isoelectric point (pI) of a given protein, the protein will assume a net negative charge. Conversely, at solution pH values less than the pI of a protein, the protein will attain a net positive charge. For unmodified nucleic acids, the low pKa of the phosphodiester backbone ensures that these molecules are negatively charged in solutions of near-neutral pH. The net positive or negative charge of a biomolecule is important because it provides a distinguishing chemical character-istic that can be used to accomplish its purification.

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Consider the following example: for a molecule with a high net positive charge, the affinity towards a cation exchange resin will be greater than a molecule with a relatively lower net positive charge (assuming the mobile phase pH is within the range to maintain a negative charge on the resin). The different binding affinities for these two hypothetical molecules can be used to accomplish their separation. If a cation exchange resin bound with both molecules is washed with buffer solutions containing increasing concentrations of a salt, such as sodium chloride, the molecule with the higher net positive charge will most usually require a buffer with a higher concentration of salt to be removed from the resin compared to the molecule with the lower net positive charge. Anion exchange chromatography exhibits analogous behavior, but instead, molecules with a greater net negative charge bind to the resin with greater affinity.

Table 1. Molecular behavior with ion exchange resins under different pH conditions

Weak/Strong Anion Exchange Weak/Strong Cation Exchange

pH above biomolecule pI Negatively charged biomolecules bind Negatively charged biomolecules do not bind

pH below biomolecule pI Positively charged biomolecules do not bind Positively charged biomolecules bind

The other determinant of affinity is the charge of the resin (Table 2), which is dependent on the pH of the mobile chromatography phase. As mentioned above, the charge of the resin can be modulated by changing the solution pH under which purification is carried out. If the pH is changed to mask the positive or negative charges on the resin, the resin will not be able to bind molecules as effectively as if the pH was in a range to leave the resin charge unperturbed.

Table 2. Behavior of anion and cation exchange resins under different pH conditions

Strong Anion Exchange Strong Cation Exchange Weak Anion Exchange Weak Cation Exchange

“High” pH(> Resin pKa)

Resin positively charged,anionic molecules bind

Resin negatively charged,cationic molecules bind

Resin not charged,molecules cannot bind

Resin negatively charged, cationic molecules bind

“Low” pH(< Resin pKa)

Resin positively charged,anionic molecules bind

Resin negatively charged,cationic molecules bind

Resin positively charged,anionic molecules bind

Resin not charged,molecules cannot bind

Practically, due to the potential tandem change in biomolecule and resin charge upon increasing or decreasing solution pH, it can be difficult to predict the binding and elution behavior of a given biomolecule with a given resin under a distinct pH condition. As a starting point, it is helpful to know the net charge of the protein or biomolecule that will be purified. If a protein is being purified, its sequence can be obtained from the NCBI database and the pI can be calcu-lated using one of many available online calculators. If an oligonucleotide is being purified, the molecule is normally negatively charged in the neutral pH regimes that DNA is normally stable under (and can become neutral or positively charged at pH ranges below 4-5). The biomolecule pI will help dictate whether to use cation exchange or anion exchange. If the pI is around 8.5 and above, then cation exchange is preferred. If the pI is at 5 or below, then anion exchange is preferred. If the pI is in the range of 5 to 8.5 then either anion exchange or cation exchange may be considered. To provide an example, if a protein pI is 6, it may be decided to perform purification around physiological pH 7.5. This means that the protein will be anionic and anion exchange can be performed. The capture buffer should have salt concentration is less than or equal to 25 mM because high salt concentrations will inhibit binding. Generally the best starting pH depends on keeping the protein stable. A solution pH that is closest to neutral but well away from the pI can be chosen to improve protein stability. At this point, two anion exchangers can be evaluated along with the best gradient.

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eA note must be made for elution techniques for ion exchange. It is not commonly known that modulation of pH can be used as well as the changing of salt concentrations for the elution of biomolecules. While salt concentration can be increased to elute proteins from strong and weak cation and anion exchange resins, pH elution is usually utilized with strong cation and anion exchange resins. The lowering of pH elutes anionic proteins from anion exchange resins, while raising pH elutes cationic proteins from cation exchange resins.

The use of ion exchange resins in laboratories is made cumbersome due to the lack of conve-nient formats to use the resins within. Commercial gravity columns exhibit a slow flow rate and the running of multiple columns and collection of fractions from the multiple columns is time-consuming and tedious. Spin column ion exchange purification is faster, but purifying multiple samples is difficult because each eluate from the spin filter must be removed and stored prior to filling the filter with the next buffer in the step gradient and centrifuging the sample. FPLC techniques overcome one major limitation of spin and gravity columns, namely the difficulty in collecting samples. FPLC typically employs a fraction collector, which separates and stores fractions for the user. A limitation of FPLC is that it is usually used for mid- to large-scale puri-fication of proteins and nucleic acids and only one column is employed at a time. This makes carrying out numerous small- to medium-scale purifications difficult because the columns are large, which dilutes the sample, and since only one sample can be purified on the in-line column, numerous samples must be processed in series, not in parallel.

Ion exchange with PureSpeed allows for the processing of multiple samples at once, while removing much of the tedium associated with ion exchange gravity and spin columns. The set up of biomolecule and buffer solutions in the deepwell plate in addition to semi-automation from the E4 XLS pipette allows for simple liquid handling of solutions over the resins within the PureSpeed tips. The eluate samples are contained within the deepwell plate, making sample collection simple. In contrast to FPLC systems, PureSpeed is able to process up to 12 samples simultaneously due to the multichannel E4 pipette. These 12 samples can be purified using any combination of strong or weak anion and cation exchange resin.

Performance of Various Ion Exchange Resins in Protein and Oligonucleotide Purification

The performance of PureSpeed Ion Exchange resins was assessed by their ability to separate oligonucleotides and proteins. For the separation of oligonucleotides (Figure 2), PureSpeed tips containing strong anion exchange resin were employed as the phosphodiester backbone of nucleic acids is negatively charged and is able to interact with the positively charged quaternary amines on the resin. A 5mer DNA oligonucleotide was mixed together with a 38mer DNA oli-gonucleotide and the two species were separated using the PureSpeed system with strong anion exchange tips. A step gradient was carried out with each step incrementally adding 25 mM of NaCl. Each eluate fraction was then scanned at 260 nm using a spectrophotometer. As it can be seen, there are two distinct peaks in the chromatogram. The initial peak is the 5mer oligo-nucleotide, while the latter peak is the 38mer oligonucleotide. These data show that the ion

Figure 2. Separation of Oligonucleotides Using Strong Anion Exchange Tips. Sixty micrograms of 5mer and 38mer oligonu-cleotides were mixed together in 500 μL of 50 mM Tris-HCl (pH 7.6) + 1 mM NaCl and sepa-rated using strong anion exchange tips. A step gradient from 1 to 725 mM NaCl was used with a step size of 25 mM. Two clear peaks are visi-ble. The first is from the 5mer oligonucleotide, while the second is due to the 38mer.

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Figure 3. Separation of BSA and Lysozyme Using Weak Anion Exchange Tips Eight hundred and thirty-three micrograms of lysozyme and BSA were mixed together in 50 mM Tris-HCl (pH 7.6) + 1 mM NaCl and separated using the weak anion exchange tips. Ten microliters of each fraction were taken, prepared into SDS-PAGE samples, then electrophoresized through 16% SDS-PAGE gels. After staining and destaining, the gels were photographed. The lane L denotes the molecular weight ladder, while M denotes the mixture of BSA and lysozyme that was separated using PureSpeed weak anion ex-change tips (these samples are included on both gels). Lane C denotes the protein sample after application to the PureSpeed weak anion exchange tip, while W indicates the fraction where the tip was washed with 50 mM Tris-HCl (pH 7.6) + 1 mM NaCl. The lanes 20 to 360 indicate eluate fractions where the NaCl concentration was increased gradually from 20 to 360 mM. As it can be seen, the smaller protein, lysozyme, elutes in the flowthrough, wash and low salt concentration eluates, while BSA (the larger protein) was re-moved from the resin between 40 and 220 mM NaCl. The reason for the different elution properties between lysozyme and BSA is because of the difference in isoelectric point between the two proteins. Lysozyme has an isoelectric point of 11.4, while BSA has an isoelectric point of 4.8.

exchange tips are capable in separating chemical species such as oligonucleotides, and likely peptides.

For protein purification, PureSpeed tips were employed to separate lysozyme and bovine serum albumin (BSA) (Figure 3). The isoelectric points of lysozyme and BSA are 11.4 and 4.8, respec-tively. This means that lysozyme is positively charged and BSA is negatively charged in neutral or near-neutral pH solutions. When a weak anion exchange resin was used to separate the two proteins, lysozyme did not associate strongly with the resin and was observed to elute in the flowthrough, wash, and early elution fractions. BSA bound to the resin and eluted from 40 mM NaCl to 220 mM NaCl.

Similar behavior was observed for lysozyme and BSA when strong anion exchange tips were used for protein separation (Figure 4). Again, BSA bound to the resin and could be eluted with higher salt concentrations than lysozyme (which again, eluted early from the resin).

Figure 4. Separation of BSA and Lysozyme Using Strong Anion Exchange Tips. Eight hundred and thirty-three micrograms of lysozyme and BSA were mixed together in 50 mM Tris-HCl (pH 7.6) + 1 mM NaCl and separated using the strong anion exchange tips. These gels were processed as per Figure 3. Lane L shows the molecular weight lad-der, while M denotes the mixture of BSA and lysozyme that was separated using PureSpeed strong anion exchange tips (these sam-ples are included on both gels). The lane labeled C denotes the protein sample after application to the PureSpeed strong anion ex-change tip, while W indicates the fraction where the tip was washed with 50 mM Tris-HCl (pH 7.6) + 1 mM NaCl. The lanes 20 to 360 show eluate fractions where the NaCl concentration was increased gradually from 20 to 360 mM. As it can be seen, the smaller protein, lysozyme, elutes in the flowthrough, wash and low salt concentration eluates, while BSA (the larger protein) was removed from the resin between 40 and 300 mM NaCl.

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eCation exchange chromatography was also employed to separate lysozyme and BSA. In cat-ion exchange, BSA is predicted to bind weakly to the resin, while lysozyme is expected to bind with higher affinity. The gel below (Figure 5), utilizing the weak cation exchange resin, shows that lysozyme is bound and purified, but it must be noted that BSA did not bind to the resin and eluted in the flowthrough, wash, and early elution fractions.

Finally, the strong cation exchange resin was used to separate lysozyme and BSA (Figure 6). Like the weak cation exchange resin, BSA did not appreciably bind the resin, while lysozyme was able to bind and be purified.

Figure 5. Separation of BSA and Lysozyme Using Weak Cation Exchange Tips Eight hundred and thirty-three micrograms of lysozyme and BSA were mixed together in 50 mM Tris-HCl (pH 7.6) + 1 mM NaCl and separated using the weak cation exchange tips. These gels were processed as per Figure 3, with the exception that only 4 μL of the BSA/lysozyme mixture was loaded. Lane L denotes the molecular weight ladder, while M denotes the mixture of BSA and lysozyme that was separated using PureSpeed weak cation exchange tips (these samples are included on both gels). Lane C denotes the pro-tein sample after application to the PureSpeed weak cation exchange tip, while W indicates the fraction where the tip was washed with 50 mM Tris-HCl (ph 7.6) + 1 mM NaCl. The lanes labeled 25 to 450 indicate eluate fractions where the NaCl concentration was increased gradually from 25 to 450 mM. As it can be seen, the larger protein, BSA, elutes in the flowthrough, wash and low salt con-centration eluates, while lysozyme (the smaller protein) was removed from the resin between 75 and 375 mM NaCl.

Figure 6. Separation of BSA and Lysozyme Using Strong Cation Exchange Tips Eight hundred and thirty-three micrograms of lysozyme and BSA were mixed together in 50 mM Tris-HCl (pH 7.6) + 1 mM NaCl and separated using the strong cation exchange tips. These gels were processed as per Figure 3, with the exception that only 4 μL of the BSA/lysozyme mixture was loaded. Lane L denotes the molecular weight ladder, while M denotes the mixture of BSA and lysozyme that was separated using PureSpeed strong cation exchange tips (these samples are included on both gels). Lane C denotes the pro-tein sample after application to the PureSpeed strong cation exchange tip, while W indicates the fraction where the tip was washed with 50 mM Tris-HCl (pH 7.6) + 1 mM NaCl. The lanes 25 to 450 indicate eluate fractions where the NaCl concentration was in-creased gradually from 25 to 450 mM. As it can be seen, the larger protein, BSA, elutes in the flowthrough, wash and low salt con-centration eluates, while lysozyme (the smaller protein) was removed from the resin between 100 and 400 mM NaCl.

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Utilization of Ion Exchange Tips in Multivariable Purification OptimizationDue to the multitude of variables that can be changed and that can affect the behavior of a protein on a given ion exchange resin, it is important to scan multiple purification conditions. PureSpeed is able to process up to 12 samples at once, so should be an excellent technol-ogy for multivariable scanning. The purification of BSA from lysozyme was analyzed chang-ing the variables of pH, salt concentration, and buffer type (Figure 7).

The multivariable optimization shows that purification cannot be carried out using citrate buf-fer, and that increasing pH in Tris buffer increases the affinity of BSA towards the strong anion exchange resin. The origin of BSA’s decreased affinity in citrate buffer is unknown, but poten-tially, this is due to citrate shielding the positively charged quaternary amine groups from BSA. The increase of BSA’s affinity as pH is increased in Tris buffer might be due to the ion-ization of additional amino acids and added negative charge. This example illustrates the power of PureSpeed in determining protein purification properties quickly, so robust protocols can more easily be developed. This experiment would be difficult to carry out with other ion exchange formats, such as FPLC, and gravity and spin columns, as they cannot process nu-merous samples in parallel.

Rainin, PureSpeed, E4, and XLS are trademarks of Rainin Instrument, LLC and Mettler-Toledo International. All rights reserved. No part of this publication may be reproduced without express written permission of Rainin Instrument, LLC. Copyright June 2013.

Figure 7. Multivariable Optimization with PureSpeed. Eight hundred and thirty-three micrograms of lysozyme and BSA were mixed together and separated using strong anion exchange tips. Different buffers of increasing pH were used in the purification. Citrate was used in the pH regimes of 4.1, 5.0, and 6.1, while Tris-HCl was used in the pH regimes of 6.9, 7.6, and 8.5. BSA was not bound in acidic conditions with citrate as the buffer, but was able to bind the resin and be purified using Tris-HCl. When the pH was increased, the elution behavior of BSA shifted significantly.

PerspectiveThe Rainin PureSpeed system functions in ion exchange chromatography and can assist a potential user due to the many options possible with the system. There are numerous benefits of ion exchange with PureSpeed. For instance:

• The PureSpeed System is validated in protein separation using strong and weak anion and cation exchange resins.

• Ion exchange with PureSpeed is flexible and can accommodate other biomolecules, such as oligonucleotides.

• With the multichannel E4 XLS pipette, up to 12 samples can be processed in parallel.• Because the PureSpeed System carries out up-and-down pipetting, there is less hands-

on time compared to manual methods.• Due to small elution volumes, PureSpeed can provide highly concentrated purified

protein.• The programmability of the E4 XLS pipette allows a researcher to easily store and trans-

fer purification protocols within a laboratory.• Multiple purification variables can be screened at once.• In conclusion, ion exchange with PureSpeed is able to yield up to 12 purified protein

samples in less time than FPLC, spin column, and gravity column techniques.

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