A number of options were explored to reduce the endotoxin level, including anion exchange column chromatography and membrane
chromatography. After small-scale laboratory work it was quickly determined that membrane chromatography technology would
offer significant advantage with regards to product recovery, time saving, and equipment requirements during manufacture.
A small-scale trial was performed using a Sartobind MA75 (2.1 mL bed volume) membrane adsorber (Sartorius). Under these conditions
the membrane adsorber provided good performance in terms of product recovery and product flow rates. From the data generated
the membrane was scaled-up for use within a 100 L scale process to a membrane bed volume of 70 mL, or a 5-in. capsule.
Before GMP manufacture, a 100 L process demonstration batch was performed to assess the feasibility of the process. First,
the fermentation broth was high-speed centrifuged to separate the cells from the basal media. The cell paste was then resuspended
in a controlled buffered solution before high-pressure homogenization. The bacterial lysate was then 0.22-μm filtered before
application to the chromatography column.
A sample of the column eluate was taken for endotoxin analysis, giving a value of 42,900 EU/mL. The bulk-process solution
was then passed through the membrane adsorption capsule with further samples being taken at 5-L intervals for endotoxin analysis
to model the capsule's clearing capacity. Once passed through the capsule, the GST-3C protease was then concentrated to 5
g/L and diafiltered into a final formulation buffer. The final product was then filtered prior to long- term storage at -70
°C. The endotoxin removal profile is shown in Figure 2.
Using the data obtained from the process demonstration batch, the membrane adsorber capsule was resized prior to the GMP manufacture
to a 20-in. (360-mL bed volume) capsule. During November 2005, two 100-L cGMP batches were performed under the same processing
conditions as the process demonstration batch, except that the column eluate was then passed through a 20-in. Sartobind SingleSep
(single use) capsule. The results from the endotoxin removal step are shown in Table 1.
Table 1. Data from the Sartobind Membrane chromatography process step from two separate batches
The use of disposable-membrane adsorption technology instead of traditional column chromatography greatly reduced plant preparation
time, because the requirement for column packing, assessment, and sanitization, was removed, along with the associated documentation,
equipment, and QC testing required to perform column chromatography. From the two batches of the cGMP manufacturing project,
a total of 415 g GST-3C protease was produced, with significant cost savings. Final product analysis can be seen in Table
Table 2. Batch data from the final product, from two separate batches
Andrew Clutterbuck is a senior purification scientist at Eden Biodesign; James Kenworthy is a purification specialist at Sartorius Biotechnology, Ltd., +44. (0).1372.737.159, firstname.lastname@example.org
and John Liddell is head of process science at Avecia.
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2. Belanich M et al. Reduction of endotoxin in a protein mixture using strong anion-exchange membrane absorption. Pharm Technol.
3. The Sartorius group. Endotoxin removal, ion exchange chromatography with Sartobind membrane adsorbers. Application note.
Gottingen, Germany: Sartorius.
4. Gottschalk U, Fischer-Früehholz S, Reif, O. Membrane adsorbers: a cutting edge process technology at the threshold. Bioprocess
Int. 2004; May;2(5):56–65.
5. Fischer-Früehholz, S. Membrane adsorbers part 1: new dimensions in chromatographic purification. GIT Lab J. 2005;2:53 –5.