Biomedical Engineering Reference
In-Depth Information
4200
Host-cell protein (ppm)
160
160
3150
900
900
600
66 0
400
400
250
1200
250
12200
2100
1600
16
116
1950
2360
1050
2850
3500
4000
0
4.5
5.1
5.8
6.4
7.0
A: Harvest pH
Figure
7.3.
The effect of harvest pH and chaotrope concentration in the rProtein A capture
column intermediate wash buffer on CHO host-cell protein (CHOP). The iso-lines represent
constant levels of host-cell protein in the rProtein A column eluate. The grayed area represents
the design space. Working within this region results in concentrations of CHOP that can be
reduced to acceptable levels in the drug substance by the subsequent purification steps.
The relationship between harvest pH and the concentration of chaotrope required for the
rPAwash step is illustrated in Fig. 7.3. Decreasing the harvest pH from7.0 to 5.8 or below
reduces the level of CHOP in the rPA column eluate from approximately 3500 ppm (ng/
mg protein) to 1600 ppm or less, even in the absence of the chaotrope wash. At this
reduced level of CHOP in the rPA eluate, the subsequent chromatography steps will
provide sufficient reduction of CHOP to achieve the required levels of residual CHOP in
the purified drug substance.
The use of the chaotrope-free process is clearly the preferred manufacturing option.
The decision was made, however, to proceed to process validation with the original
process and include the improved process as an alternate harvest-capture protocol in the
initial biological license application. Obtaining regulatory approval of the alternate
harvest-capture protocol prior to implementation will minimize the regulatory risks
associated with making process changes late in clinical development.
7.5 PROTEIN A CAPTURE COLUMN
Protein A resins exhibit relatively low binding capacity (25-40 g product/L of resin)
and thus require a large column or a smaller column with multiple chromatography
cycles to capture the entire amount of product produced in cell culture. This is of
