A Universal Method for Basic cIEF

The Application Notebook

The Application Notebook, The Application Notebook-02-01-2009, Volume 0, Issue 0

Achieve highly reproducible cIEF separations of basic mAbs using the basic pH gradient cIEF separation method on the PA 800 Protein Characterization System.

Achieve highly reproducible cIEF separations of basic mAbs using the basic pH gradient cIEF separation method on the PA 800 Protein Characterization System.

The use of cIEF for the characterization of therapeutic monoclonal antibodies (mAbs) has been increasingly adopted in recent years. The pl determination adds a critical dimension in establishing identity, purity, post-translational modifications, and stability of therapeutic mAb preparations. Most mAbs have charge isoforms with a pl in the basic range of the pH gradient (7–10). However, cIEF in this region presents a challenge due to presence of "poor" carrier ampholytes in the basic pH region and decay of the gradient by isotachophoresis. This application note describes a robust, high resolution cIEF method that incorporates anodic and cathodic stabilizers.

Experimental Conditions

Table I: Master mix preparation

A master separation mix was prepared and 10 μL of 5 mg/mL mAb was added to 240 μL of the master mix.

Table II: Instrument setup

Results

Electropherograms of three basic mAbs were separated using the same cIEF conditions, resulting in highly unique peak profiles and detection times (Figure 1). The unique peak profile for different mAbs shows that a single method using a broad pH 3 to 10 ampholyte mixture is capable of resolving differences in pl up to a few hundredths of 1 pH unit across the basic range of the gradient.

Figure 1

Conclusions

A single separation method can be employed to perform cIEF analysis of multiple mAbs across a broad pH range. Workflow is greatly simplified by the use of a platform method, reducing the chances for operator error and increasing overall efficiency in method development. The resulting reduction or elimination of method optimization also allows for easier scale up of cIEF analysis in routine use environments. Statistical analysis of pl and isoform group percent composition confirms that highly reproducible cIEF separations of mAbs can be achieved even in the previously problematic basic region of the pH gradient.

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