How do Molecular Attributes and Sample Properties Affect Chromatography?

Discover How the Features of Your Biomolecule Will Shape Your Downstream Purification Strategy.

This article is posted on our Science Snippets Blog 

affinity chromatography


It can sometimes feel as though new chromatography matrixes or functionalities pop up every year. This progress is undoubtedly impressive but can make choosing the right chromatography method for your own application difficult. Should you use tried and true resins? Should you opt for a more modern matrix? More importantly, how do you decide?

While the choice can be difficult at first glance, there is logic to finding the right approach. You start by looking into the molecular attributes of your desired product and the impurities that must be removed for its successful manufacture.


The more you understand the properties of your starting material and product, the easier it will be to find the right chromatography technique for your purpose.


In this article, we review how different molecular attributes and feed properties affect separation capabilities and guide you towards the best technique for your application. This will not be an exhaustive guide to different chromatography methods but will instead highlight some principles that you can use to judge the appropriateness of any technique for separating therapeutic biomolecules.


Properties Influencing all Chromatography Methods

In chromatography, there are two sets of properties that influence your initial purification choices:

  1. The molecular attributes of your target molecule
  2. The molecular attributes of every impurity and contaminant that you wish to remove

Looking closer at the molecular properties of a sample, you will find many features that are useful for separation using different types of chromatography (see Tables 1-3). Differences in size, charge, hydrophobicity, solubility, and H-bonding energy can all deliver excellent separation of impurities and targets. Likewise, a difference in affinity of a capture ligand for the target versus impurities can provide a very efficient separation step or unit operation.


chromatography columns


Ideally, you will be able to identify big differences in the molecular properties of your target molecule and the impurities, making it easy to separate them. A thorough analysis of your target, impurities, and potential contaminants forms the basis for the initial screening of candidate matrices and separation modes.

At this point, some approaches can be excluded, and specialized methods might be considered for the most challenging separations. For example, the existence of an appropriate affinity chromatography (AC) matrix might be a good choice for an early target capture step. Classic examples use a Protein A ligand for IgG and an Oligo dT ligand for messenger RNA (mRNA). However, using an affinity adsorbent might not solve issues like the removal of molecules similar to the target (those that carry the same affinity binding site but differ in other ways). Such challenges are usually handled later in a purification scheme, in what are called polishing steps. Examples include the removal of insulin variants by reversed-phase chromatography (RPC) and the separation of antibody aggregates by mixed-mode chromatography (MMC).


Thorough Knowledge of Your Sample Will Help Optimize the Chromatography Workflow

Understanding your sample, feed, or starting material is essential. Typically, scientists will have a lot of information about the purification target. You likely know the isoelectric point and the amino acid composition if you work with proteins, or the nucleic acid sequence if you work with DNA or RNA (Table 1).


Table 1. Properties of your target.

Property

Impact

Comment

Large molecule target

Use convective chromatography devices (monoliths or membranes).

Beaded resins have a low capacity for large biomolecules due to diffusion limitations and require low flow rates. Note mRNA and plasmid DNA (pDNA) molecules are much larger than the proteins they encode.

Low concentration target

Bind target, not impurities. Elute with a step gradient.

Commonly ion-exchange chromatography (IEX), affinity chromatography (AC), or hydrophobic interaction chromatography (HIC).

High concentration target

Can use target flow-through and bind impurities (negative chromatography).

Commonly, IEX is used. Can be very fast. Regeneration and cleaning of the matrix will be essential if the device is re-used.

Charge differences between target and impurity

Use IEX.

Broadly applicable. Large range of matrixes available and many method options.


You’ll know if your target is fragile and easily damaged by shear or if it can survive harsh mechanical treatments. You’ll have data on the hydrophobicity and a whole collection of data on stability and activity under different conditions (Table 2).


Table 2. Reducing damage to your target

Property

Impact

Comment

Shear sensitivity

Avoid beaded resin matrices, use monoliths.

Allow high flow rates without turbulence.

Binding site

Potential for rapid AC with a highly selective ligand.

For example, Oligo dT for mRNA, Protein A for IgG. Efficient, robust platform approaches are available. Will usually reduce the number of purification steps. Even possible to remove a specific impurity by AC.

Degrading enzymes

Remove early, for example, in the capture stage. Use high flow rates.

Could include proteases, nucleases, and other modifying enzymes.


Data on the impurities in your sample is often less abundant. Collecting this data gives you an excellent opportunity to thoroughly compare the molecular attributes of your target to the impurities to help find the best strategy for purification (Table 3).


Table 3. Removing impurities

Property

Impact

Comment

Charge differences between target and impurity

Use IEX.

Broadly applicable. Large range of matrixes available and many method options.

Hydrophobic differences between target and impurities

Use HIC.

Method development can be complicated. Cleaning the matrix for re-use may also be challenging.

H-bonding energy

Often overlooked due to weaker bond strength. Consider suitable MMC matrix.

Works at higher salt concentrations and allows simultaneous sorting based on size.

Target and impurity with similar charges

Consider changing pH.

Often limited by unstable components.

Target and impurities closely similar

Address in polishing stage. Use high-resolution matrices.

Examples include MMC for IgG and reversed-phase chromatography (RPC) for insulin variants. Might require a shallow gradient and finely beaded resin.

Target much larger than impurities

Size-exclusion chromatography.

Target flows through. Impurities penetrate beads and bind. Low capacity can be impractical.



Chromatography Process Considerations

The end result of any commercial chromatography process has to be a product with the right critical quality attributes (CQAs). These are the predetermined properties of the target molecule that make it fit for purpose. Sometimes this is called the target product profile (TPP). The activity of the target is clearly important. In an ideal case, you will understand the molecular attributes responsible for this activity and the conditions that preserve or disrupt these attributes during chromatography.


Even with high recoveries of, let’s say, 90% per step, a four-step process results in an overall yield of around 66%.


 pH

The different chromatography steps needed to achieve a pure product will impact yield because special conditions demanded by the technique might damage the sample components in some way. Very high or low pH - useful for modifying the charge on proteins for IEX - could easily lead to protein denaturation and irrecoverable activity loss. Some proteins are particularly unstable at their isoelectric points, where they carry no net charge and can easily precipitate. Damaging the product in such ways is clearly not ideal. Damaging the impurities might be one way of getting rid of them, but just as often, it could complicate your purification challenge.


Ionic Strength & Conductivity

Similar to considering the impact of operating pH on yield, it might be necessary to operate within a range of ionic strengths (often monitored as conductivity). Taken together, one can visualize the working ranges of pH and ionic strength as a stability window – an area within which the target is stable. In most cases, it is preferred that the impurities are also stable (unless they actually damage the target) to simplify run-to-run consistency.

Ionic strength, of course, has a direct impact on the performance of many modes of chromatography, in particular IEX and HIC, but also MMC and AC.


Physical Characteristics of the Chromatography Matrix

The physical characteristics of a chromatography matrix can also impact the stability of molecules being separated and, consequently, yields. For example, large shear forces can be disruptive for long and delicate molecules like mRNA. Flow around resin beads packed in a column is often turbulent, adding to shear forces. In contrast, flow through a monolith, with its uniform, interconnected channels, is usually laminar, making this format more appropriate for large, shear-sensitive molecules. Turbulence in a column packed with resin beads will also impact resolution negatively.


Speed

Sometimes, the order of steps in a purification workflow and the speed of a particular chromatography step will strongly impact target stability. Impurities that will destabilize your target if given enough time are often present, for example, proteases that digest protein targets. It is essential to inactivate and remove these impurities quickly - as early as possible in the purification scheme and with a fast method - to protect your target and maintain high activities and yields.


Example 1: mRNA

Figure 1 – Monolithic chromatography (CIMmultus™ Oligo dT) can be used to effectively separate mRNA molecules from non-polyadenylated species.



mRNA is an example of a molecule that is being used increasingly in biotherapeutics. Several fundamental properties dictate the purification of mRNA. The first is a unique chemical affinity, the second is the charge, and the third is hydrophobicity. All mRNA molecules also have a unique signature in the form of a poly-A tail, which consists of several adenosine residues tagged to the end of the mRNA when it is synthesized. This unique property provides a way to separate mRNA using an affinity matrix with a string of complementary bases, i.e., oligo dT molecules attached to a chromatography matrix. All nucleic acids are negatively charged and can easily be separated from positively charged impurities using an ion exchanger. HIC is also commonly used when purifying mRNA, especially when they do not possess a poly-A tail.  

Often a healthy mix of AC, IEX, and HIC is used to purify mRNA. Overall, these unique properties provide a relatively straightforward approach to purifying mRNA for use in therapeutics such as vaccines.


Example 2: Proteins  

Proteins are often more complex to isolate than nucleic acids because they are expressed in cells containing many other proteins with a variety of similar characteristics. The purification process must be capable of separating the target protein from similar cellular proteins. The following molecular attributes are commonly used as a basis for separation:

  • Proteins usually carry different charges, meaning you can separate them on an ion exchanger. Different Isoelectric points allow you to differentiate your target protein from the other protein impurities. Sometimes a change in pH can enable the removal of key impurities by changing their charge properties and allowing a previously impossible separation by IEX.
  • Proteins can be separated based on their hydrophobicity using hydrophobic interaction chromatography.
  • Many proteins also have high affinities for specific molecules. For example, Protein A binds strongly to the Fc portion of antibodies, making it an excellent candidate for separating an antibody target, such as a monoclonal antibody (mAb), from other cellular components.



Number of Chromatography Steps and Process Yield

It is typical that purification of a biotherapeutic will require two, three, or more different chromatography steps.


Even with high recoveries of, let’s say, 90% per step, a four-step process results in an overall yield of around 66%.


This simple calculation shows you how important it is to reduce the number of steps in a purification process, rather than just adding steps until the target is sufficiently pure. Understanding attributes of the target modality and impurities will help you find the methods that will most effectively purify your product in the fewest number of steps. This achieves not only a suitably pure product but can also have a significant impact on costs, throughput, and overall production yield.


Summary

Chromatography methods for bioprocessing are numerous and wide-ranging. However, truly understanding the properties of your sample - including the molecular attributes of both the purification target and the impurities - can help streamline the selection process. This includes finding the best methods for your project, getting the order right, and minimizing the number of processing steps in your chromatography workflow. 


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