While typical freeze-dried samples did not contain detectable dimer, the mannitol-only formulation (Figure 3) was used to
produce a dimer-containing sample for SEC method development. Initially, a Zorbax GF450 column was used with 0.2 M sodium
phosphate pH 7.0. Samples were diluted with the mobile phase and the method was run isocratically at a flow rate of 1 mL/min.
When using the pH 7.0 mobile phase, a dimer peak was not observed. However, when the mobile phase was adjusted to pH 8.0,
the dimer peak was visible but not well resolved from the main peak.
To improve resolution, we tried a Phenomenex BioSEP-SEC S-3000 column, and to simplify mobile phase preparation, a commercially
prepared phosphate buffered saline (PBS) pH 7.4. The dimer was observed and resolution was significantly improved. The limit
of quantitation for protein aggregates was less than 1 percent, which is significantly better than that for SDS-PAGE.
A stability-indicating chromatographic method was required to provide product concentration and purity data for further formulation
development, product release testing, and stability testing. The RPC potency method initially provided by the bulk manufacturer
was evaluated. This method used a Poros R2 column from Applied Biosystems with a NaOH-based mobile phase. Samples with degradation
products were prepared by heating a sample of reference standard solution at 37°C for 12 hr. Despite changes in flow rate
and gradient, degradation products could not be resolved from the main peak using the bulk manufacturer's conditions.
Because of the large size of the protein, we evaluated a Toso Biosep TSKGel Phenyl 5PW RP 1000A pore size column. We also
analyzed mobile phases such as NaOH, sodium phosphate, Tris, 0.1 percent HCl, and 0.1 percent TFA. Optimized method conditions
used a mobile phase A of 0.1 percent TFA in water, mobile phase B of 0.1 percent TFA in ACN, and a column temperature of 35°C.
The optimized RPC method was capable of partially resolving dimer as well as clipped product (Figure 4).
Figure 4. Reverse-Phase Purity and Potency Method. Samples include a control (1), a sample containing aggregate generated
by freeze-drying in only mannitol (2), and a sample of API with a truncation (3).
IDENTITY TESTING BY WESTERN BLOT
FDA specifies that identification procedures be specific for the active ingredient whenever possible.8 Western blotting was selected as an identity method because it identifies the product both by molecular weight and by detection
with a product-specific monoclonal antibody.
The western blot used the same SDS-PAGE materials described above, as well as NuPAGE transfer buffer and membranes from Invitrogen.
The membranes were immunostained by first incubating them in a blocking buffer of non-fat dry milk in PBS, followed by the
primary antibody, followed by a phosphotase-conjugated secondary antibody, followed by a colorimetric substrate, BCIP-NBT.
During method development, both a polyvinyldene fluoride (PVDF) and a nitrocellulose membrane were evaluated with sample loads
of 0.1, 0.2, and 0.4 µg. The PVDF membrane had high background staining and was not used in further method development. All
of the tested sample loads resulted in acceptable bands, and the 0.2 µg level was used for further testing. The identity method
was validated for specificity and transferred to the quality control laboratory for product release testing.
PRODUCT RELEASE ASSAYS
Methods based on seven different techniques were developed for support of formulation development and for validation as product
release assays (Table 1). Four were validated and transferred to the quality control laboratory. RPC, SEC, and WAX chromatographic
methods were developed and ultimately used for product release in place of SDS-PAGE and IEF because the methods were more
robust and results were easier to interpret.
Table 1. Methods Summary
Though not used for final release, electrophoresis was an invaluable first screening tool. Because SDS-PAGE and IEF required
minimal development time, they provided the formulation development scientist with immediate methods to assess product quality.
The observation of dimer by SDS-PAGE was used to eliminate a destabilizing formulation early in development. IEF, though a
more difficult technique, provided the protein's isolectric point and a means of evaluating charge variation that was further
investigated by anion-exchange chromatography.
Wendy Saffell-Clemmer is manager of analytical development, Pharmaceutical Research and Development, Baxter Pharmaceutical Solutions LLC, 927 S. Curry Pike, PO Box 3068, Bloomington, IN 47402-3068, 812.355.7105, fax 812.332.3079.
1. Roth G, Parenteral Sector Watch, Contract Pharma. 2003 November/December. Available at: http://