Process Resins - Ion Exchange

1. What is the difference between a strong ion exchanger and a weak ion exchanger? How do I know which one to choose for a particular application?

The terms “strong” and “weak” refer to the acid/base properties of the functional group. If the ligand is derived from a strong acid or a strong base it is referred to as a strong ion exchange resin. If the ligand is derived from a weak acid or a weak base it is referred to as a weak ion exchange resin. For example, TOYOPEARL SuperQ-650 resin (pKa about 12.2) is functionalized with a quaternary amine which is considered a strong base. Hence it is called a strong anion exchange resin.

However, TOYOPEARL DEAE-650 is functionalized with a putative weak basic group and is called a weak anion exchange resin. The pKa of TOYOPEARL DEAE-650 is approximately 11.5 which is quite high for a weak base and is therefore amenable to buffers much higher than other DEAE resins. The same is analogous for cation exchange media.

In either case the interaction is based on ionic interactions and the strength of binding (unless very close to the pKa) is not considered strong or weak but is similar for any ion exchangers. For most chromatography applications, the operational pH range for proteins is between pH 5.0 and 9.0, so either resin may be used.

2. How can I maximize resolution between my target molecule and impurities when using Ion Exchange?

There are many ways in which better separation can be achieved in ion exchange chromatography and these guidelines can be applied to other modes of chromatography as well. First, resolution can be maximized by increasing bed height. Typical column bed heights range from 15 cm to 30 cm L. Going any higher than 30 cm will result in higher back pressure and reduced throughput if the linear velocity needs to be reduced. A benefit of greater bed heights is to increase sample residence time in the column at the same flow rate, resulting in an increase in binding capacity. Second, a gradient may be used instead of step elutions to resolve impurities. The slope of the gradient can be adjusted to provide either a sharp or gradual change in conductivity depending on how close the impurities elute to the target molecule.

Be aware that shallow gradients result in broad elution peaks. Lastly, pH can be adjusted to affect how tightly molecules bind and subsequently elute from the column. Small changes in pH can cause retention time shifts for elution peaks and may allow for better separation. Furthermore, if the pKa of the target molecule is vastly different from contaminants, either anion or cation exchange may be appropriate if the pH is adjusted above or below the pKa of the target molecule.

3. In what cases is Ion Exchange used in protein purification?

Ion exchange is commonly used in capturing and concentrating target proteins (since all proteins have some charge associated with their primary structure) from fermentation feedstocks. Ion Exchange is also used in intermediate steps to purify aggregates and charge variants. 

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