BIOPHARMACEUTICAL MANUFACTURING - Risk Management Applications in Pharmaceutical and Biopharmaceutical Manufacturing - page 334

The choice of a recovery/purification process is typically based on
process/product-specific criteria such as the intracellular or extracellular location
of the product, the concentration of the product in the harvest fluid, the minimal
acceptable standard of purity, facility limitations, etc. Detailed information on
downstream processing can be found in R. Bates [17].
Microbial Products For fermentation or microbial products (i.e., obtained from
bacteria, yeast, and fungi) that are usually intracellular, the recovery and purifi-
cation process can be summarized as follows: the first step is the removal of
large solid particles and microbial cells. The method commonly employed for
this purpose is either filtration or centrifugation. In the next step, the cells are
lysed either by chemical or mechanical methods to release the product. The cell
lysate is then fractionated (primary isolation of the product) using techniques
such as ultrafiltration, reverse osmosis, adsorption/ion exchange/gel filtration, or
affinity chromatography. After this step, the product-containing fraction is further
purified by fractional precipitation, or more precise chromatography techniques
(intermediate purification/concentration) to obtain a product that is highly con-
centrated and essentially free from impurities and ready for filtration and fill
[18]. The flowchart in Figure 12.6 depicts the typical stages of extraction and
purification of fermentation products.
Mammalian Products For therapeutic proteins (i.e., obtained from mammalian
cells) that are usually extracellular and secreted, the recovery and purification
process are briefly described as follows: the first step is the clarification and ini-
tial purification stage. The main goal of this step is to reduce the working volume
of the process stream and to remove the cell debris and harmful contaminants.
The speed of recovery is critical in this step to minimize degradation and product
loss. Typical unit operations include the use of filtration (including depth filtra-
tion to reduce solids content and ultrafiltration and diafiltration to reduce volume
and change buffering conditions), chromatography, precipitation, or centrifuga-
tion techniques. In the next step, once the whole cells are separated from the
product-containing harvested cell culture fluid (HCCF) during clarification, the
HCCF can often be applied directly onto a chromatographic column for initial
purification. Typically, filtration, centrifugation, ion exchange column (IEC), or
hydrophobic interaction columns (HIC) are used for this purpose. Ideally, this
initial purification results in a substantial reduction in the level of proteases or
other harmful components. In the next step, intermediate processing, most of
the remaining contaminants are removed from the process stream (e.g., virus
particles, nucleic acids, endotoxins, and other host cell proteins). Unit opera-
tions with a large capacity, high recovery, and good resolving power such as
adsorption chromatography (e.g., ion exchange, hydrophobic interaction, affinity
chromatography, etc.) and membrane chromatography are common methods for
use in this stage. The final step of the process is concentration and polishing.
This step removes impurities such as deamidated isoforms or aggregates using
chromatographic techniques such as gel permeation chromatography (GPC). In