Orthogonal Chromatographic Techniques for Therapeutic Protein Characterization

1st March 2018

Category: Bioanalytical

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By: Stuart Wright - Scientist,

Development of biotherapeutics, for example monoclonal antibodies (mAbs), requires a wide range of physicochemical and structural analysis to be undertaken in accordance with ICH Q6B guidelines. Each technique will assess different critical quality attributes (CQAs) which form part of the analytical characterization of the biotherapeutic.

CQAs are properties or characteristics relating to physical, chemical, biological or microbiological functions of a biotherapeutic. CQAs are required to be closely monitored during a biotherapeutic’s production to ensure they are within a defined range or limit, which in turn ensures the product is of sufficient quality and safety.

Three chromatographic techniques with orthogonal selectivities that can be utilized to assess different CQAs are:

  • Size Exclusion Chromatography (SEC)
  • Ion Exchange Chromatography (IEX)
  • Reversed-Phase (RP) Chromatography

Size Exclusion Chromatography (SEC)

Size exclusion chromatography is a technique for the detection and quantification of soluble aggregates and fragments of biological molecules. The presence of aggregate species in biotherapeutics is known to affect safety and efficacy. It is therefore important to monitor the amount of aggregation during the production of a biotherapeutic.

Separation in SEC is based on the size of products present in a sample and their ability to move through a column made up of an inert stationary phase with pores of a defined size. Smaller molecules pass more deeply into the pores compared to larger molecules. Therefore, the smaller molecules take a longer path to get through the column and are separated from the larger molecules.

Figure 1 shows comparison data between a monoclonal antibody and the same mAb after being exposed to differing amounts of heat stress. Each chromatogram shows 1 main peak, 2 high molecular weight peaks and 2 low molecular weight peaks. The high molecular weight components increase in intensity with increased exposure to heat stress. Aggregation is a common degradation pathway for therapeutic mAbs and so, SEC is an important tool when assessing a biotherapeutics stability because of its ability to resolve these impurities.

Sartorius Stedim BioOutsource blog

Figure 1. A comparison of a sample after being put under different degrees of stress at 37oC. Unstressed (blue), 18 hours of heat stress (red), 90 hours of heat stress (green) and 1 week of heat stress (black).

Ion Exchange Chromatography (IEX)

Ion exchange chromatography is a technique for the detection and characterization of charge variants of biological molecules. Charge variant analysis is important in the characterization of biotherapeutics because it provides information about product quality and stability. Charge variants can be caused by modifications, such as C-terminal lysine truncation, deamidation and other post-translational modification. Separation occurs on an ion exchange column. One possible approach is to use pH gradients: this is shown in the example below (Figure 2).

Sartorius Stedim BioOutsource chromatographic blogFigure 2. A comparison of charge variants of an innovator (black) and a biosimilar (red) mAb using ion exchange chromatography.

Figure 2 shows a comparison between an innovator mAb and a biosimilar mAb. The main chromatographic peaks are present in both, however, they are present in different quantities. Due to the samples being separated using cation exchange, the most acidic components have eluted first and the more basic components have eluted last. Comparison of the chromatograms shows that the biosimilar contains higher levels of acidic species. A structure-function study would be required to determine the biological impact of these differences.

Reversed-Phase (RP) Chromatography

Reversed-phase chromatography is a widely used analytical technique for the characterization of biological molecules. Separation is based on hydrophobicity. Minor changes in a biotherapeutic molecules structure can have an impact on its overall hydrophobicity. For example, IgG2 molecules have different disulfide bond structures, which can be resolved by RP chromatography. In addition, oxidized impurities exhibit different RP retention times (Figure 3).

Sartorius Stedim BioOutsource chromatgraphic blog graph

Figure 3. A Reversed-Phase chromatogram of an intact IgG1 mAb (black) and the same mAb after undergoing oxidation from reaction with hydrogen peroxide (red).

Figure 3 shows a comparison between an intact IgG1 mAb (black) and the same mAb after undergoing oxidation due to reaction with hydrogen peroxide (red). The oxidized mAb elutes earlier in the chromatogram. Oxidation of biotherapeutics, such as mAbs, can have an adverse effect on shelf life and bioactivity. These data demonstrate that RP Chromatography is an effective technique for detecting oxidized impurities from a biotherapeutic sample.


The three chromatographic techniques outlined above show that different CQAs can be analyzed using robust liquid chromatographic techniques when performing protein characterization. When analyzing CQAs of biotherapeutics, it is important to use orthogonal techniques to gain as great an understanding as possible of the various structural properties. For all therapeutic proteins, robust analytical assessment of CQAs allows for better control of the manufacturing process. For biosimilars, detecting differences relative to the reference molecule early in the development process drives decision-making and enables process optimization to be performed for successful market approval.

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