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Intermediate Purification
In the Intermediate Purification phase the focus is to separate the target protein from remaining impurities such as other proteins, nucleic acids, endotoxins and viruses.
After capture, the feed is less crude and more consistent. The impurities to be removed are more defined and screening can include specific analyses. In Intermediate Purification, ability to resolve similar components is of increased importance. Specific "regulatory contaminants" must be removed.
Medium bead-size media are usually used to improve resolution and in some cases linear gradients can be applied. Capacity will still be important to maintain productivity. Speed is less critical in intermediate purification since the impurities causing proteolysis or other destructive effects should have been removed, and sample volume should have been reduced.
Filtration techniques like UF or DF are useful for conditioning the feed before Intermediate Purification. | |
Intermediate Problem
Technique | | | |
| High ionic strength, capture step was IEC | Yes,
IEX media with complementary selectivity and may be condition sample by diafiltration | Best,
if target molecule binds to HIC media (FF or HP) | Yes,
for buffer exchange only |
| Low ionic strength, capture step was HIC | Best,
using FF or HP media | Avoid,
or use HP media only | Avoid |
| High DNA/endotoxin content | Yes,
use cation exchanger to bind target molecule or anion axchanger in negative mode | Yes,
alternative to IEC | Yes,
in group separation |
| Contribution to virus removal | Yes,
at low pH if compatible for target molecule, alternatively use solvent-detergent technology | Yes,
alternative to IEC or use solvent-detergent technology | Yes,
if sample volume is small, or use solvent-detergent technology |
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