Biology Reference
In-Depth Information
range between 1,000 L and 20,000 L. To harvest the product, the cells must be
separated from the liquid medium. Separation is achieved by microfiltration or
centrifugation. The clarified liquid-containing product is then concentrated for
volume reduction and purification.
Within any of the process modes described above, a cell line that has been
engineered to produce rHuEPO may grow and secrete product encoded by the
EPO
gene. Not all of the secreted product may qualify as the rHuEPO sold by
a manufacturer. The EPO molecule has three
N
-glycosylation sites and one
N
O
-glycosylation site, leading to the possibility of differently charged mole-
cules with different isoelectric points (i.e., isoforms). Those molecules with
the lowest isoelectric points, typically corresponding to those having the great-
est amount of sialylation and glycosylation branching, have the greatest
in vivo
efficacy. Molecules with higher isoelectric points, or less sialylation and less
glycosylation, have less therapeutic efficacy, and can be removed in purifica-
tion. (See Chapter 13 for further information about isoforms.) Additionally,
even if the cell produces product of the desired charge profile, this product
may be degraded in culture by proteases. The product itself may form unde-
sired complexes such as aggregates, which must be separated from the desired
monomer. The cell may be influenced to produce increasing amounts of the
desired charge profile by controlling process conditions, such as temperature,
pH, osmolality, and nutrient and waste product concentrations. Similarly,
undesired forms of the product may also be minimized.
Recovery and purification of erythropoietin
A number of purity criteria must be met for rHuEPO to be a viable human
pharmaceutical. Host cell and other contaminants, such as DNA, host cell pro-
teins, and endotoxin, must be removed to appropriate levels. The removal or
inactivation of any theoretical endogenous or adventitious viruses must be
shown. Amounts of product-related contaminants, such as aggregates, prote-
olytically degraded,under-glycosylated, or oxidized forms, are minimized. A
targeted purity is specified by the manufacturer, so each product lot must meet
the criteria described in the manufacturer's product specification. Product
specifications may differ among manufacturers.
The clearance rate of rHuEPO in the human body is determined by the num-
ber of sialic acid residues on the carbohydrate portion of the molecule. Under-
sialylated rHuEPO are cleared in the liver by the asialoglycoprotein receptor
[5]. Selecting for molecules with the greatest degree of sialylation gives a
product with the desired pharmacokinetic properties, which affect the biolog-
ical potency of rHuEPO. Different manufacturers may have different selection
criteria (and therefore different product quality characteristics) for the EPO
molecules that are retained in the recovery and purification processes.
In addition to having a well understood and reproducible recovery process,
a number of regulatory requirements exist for the manufacture of biopharma-
