A Case Study of Developing Analytical Methods - A contract manufacturer used seven analytical methods while developing an early-phase protein therapeutic in six short months. - BioPharm International


A Case Study of Developing Analytical Methods
A contract manufacturer used seven analytical methods while developing an early-phase protein therapeutic in six short months.

BioPharm International

The first IEF test used materials purchased from Invitrogen because these were available in the laboratory. An API sample was desalted using a 5K molecular-weight-cut-off spin filter. The sample was diluted in pH 3-10 IEF sample buffer and then samples and pI markers were loaded on a pH 3-10 IEF gel. Three major, poorly resolved bands migrated between the pH 5.3 and 6.0 markers.

Because multiple bands were observed, a method for further characterization was required to ensure that formulation and process conditions were not affecting the pattern of charge variants. The IEF bands' sharpness and resolution were improved by using a larger gel on a flat-bed system. A recirculating chiller assured temperature control. Flat-bed IEF was performed using equipment and materials purchased from Amersham Biosciences. An Ampholine PAGEplate pH 3.5-9.5 gel was used, with 1M phosphoric acid as the anode buffer and 1M NaOH as the cathode buffer. After staining with Coomassie blue, the gels were placed on a white-light transilluminator and photographed with the Kodak EDAS 290 system.

Resolution was better. Six bands, which migrated between the pH 5.2 and 5.85 markers, were resolved. Because the protein was not glycosolated and had no disulfide bonds, the charge variants observed by IEF were the major forms of molecular heterogeneity requiring characterization.

Figure 1. IEF of Reference Standard (1), API (2) and Final Product (3).
IEF was used throughout the development process to compare lots of API, compare API to final product (Figure 1), and determine reconstitution stability under different conditions. IEF required little development time and was an excellent tool for visually comparing samples within one gel, but intermediate precision was poor. We observed changes in the reference standard's band pattern after a new lot of gels was used during the course of the project. Although densitometry could be applied to IEF gels, changes in the band pattern could not be easily quantitated, making IEF a poor choice as a product release method in the quality control laboratories.

ION-EXCHANGE CHROMATOGRAPHY We searched for a chromatographic method to release product and track changes in product stability samples. Ion-exchange chromatography separates proteins on the basis of charge through the interaction of the protein with oppositely charged groups immobilized within a column.5 When the mobile-phase pH is greater than the protein pI, the protein has a net negative charge and binds to positively charged anion-exchange columns. Conversely, when the mobile phase pH is less than the protein pI, the protein will bind to negatively charged groups in a cation-exchange column.

We evaluated both anion-exchange chromatography and cation-exchange chromatography. Because of previous experience in evaluating charge variants of monoclonal antibodies, we analyzed a cation-exchange method using a Dionex weak-cation-exchange (WCX)-10 column with sodium phosphate mobile phase. The protein was eluted with an increasing gradient of sodium chloride in 50 mM sodium phosphate. Mobile phases with pHs from 5 to 6 were analyzed, with the best peak shape obtained at pH 5.3. The main peak tailed considerably and only two additional peaks were observed, both poorly resolved.

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