Quality by Design in the CMO Environment - - BioPharm International


Quality by Design in the CMO Environment

BioPharm International
Volume 20, Issue 12


Figure 2
The HIC resin used for this case study was Octyl Sepharose 4 Fast Flow (GE Healthcare, catalog number 17-0946). Octyl Sepharose demonstrated separation of different species with a reverse salt gradient. Protein A capture of the antibody produced a partially purified feed stream with three major impurities, referred to in this paper as species A, B, and C. Figure 1 shows size-exclusion high-performance liquid chromatography (SEC-HPLC) of a side fraction from the Octyl column enriched for the three product variants. On Octyl sepharose, species B co-elutes with the beginning of the product peak, and species A and C elute toward the tail of the product peak (Figure 2). Although there is overlap, initial screening experiments indicated that certain conditions increased the resolution between the species.

Table 1
The DOE looked at six parameters: column bed height, protein loading density, pH, temperature, flow rate, and salt concentration of the load. Dependent variables analyzed were yield, purity, elution gate, and resolution between product and impurity peaks. JMP 6 software (SAS Institute) was used to design the experiments and analyze the data. Because of the number of variables (6) and limited material and time, a fractional factorial design with two center points was chosen (Table 1). It is sufficient to identify main effects and some two-factor interactions.

Three 1.0-cm diameter jacketed columns were packed with Octyl resin to represent a tall (27.2 cm), short (14.6 cm), and center-point (22.5 cm) bed height. Packing efficiency tests were run on the columns to ensure consistency between the columns. The columns were sanitized with 0.5 M NaOH and stored in 20% ethanol before the first run and after each run to minimize the possibility of column fouling. Equilibration and elution buffers were prepared at pH 6.0, 6.5, and 7.0.

Water baths were set up to recirculate water at the specified temperature through the jacket of the column. In addition, a stainless steel coil was plumbed to the inlet of the column and submerged in a jacketed beaker of water, also connected to the recirculating bath to ensure temperature control. The chromatography systems used were GE Healthcare ÄKTA Explorers with inline flow restrictors.

Load material was produced by processing clarified harvest material from two 10-L cultures over a Protein A capture column. The Protein A eluate was adjusted to pH 6.0 and frozen in aliquots at –70 °C. For each chromatography run, thawed Protein A eluate was filtered and diluted to the appropriate ammonium sulfate concentration with 2 M (NH4)2SO4 at pH 6.0, 6.5, or 7.0. The protein solution was filtered again and UV absorbance at 280 nm was measured to calculate the total protein concentration of the the load volume required for each run. The load was equilibrated in the water bath to attain the specified temperature before starting the chromatography run.

Table 2
Each run was performed using the method outlined in Table 2 while incorporating the specified pH, temperature, and flow rate parameters from the DOE scheme (Table 1). One column-volume (CV) fractions were collected throughout the elution with an automated fraction collector. Absorbance at 280 nm was used to determine concentration, and SEC-HPLC was used to determine purity for each fraction.

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