Biology Reference
In-Depth Information
ceuticals. The product must be produced under Good Manufacturing Practices
(GMP), which encompasses a range of topics from plant design, which affects
air and water quality,solvent usage, and waste disposal, to day-to-day opera-
tions, which include operator training, batch record writing and review, quali-
ty assurance, and cleaning validation. Many of these guidelines can be found
in “Q7A Good Manufacturing Practice Guidance for Active Pharmaceutical
Ingredients” [6].
Concentration and diafiltration
After the clarified cell culture media is harvested, it must be concentrated, and
the buffer salts and other high conductivity/low-molecular-weight components
must be removed. Concentration is an important step, since it is difficult to
store and handle large-pool volumes. The expense of storing large volumes of
harvest media in a frozen state can be prohibitive. In addition, the time
required to load a large volume of media on to a column decreases plant pro-
ductivity. Prolonged column load times can also lead to proteolytic degrada-
tion of product in the harvest media as it awaits loading onto the column.
Removal of buffer salts and other high-conductivity, low-molecular-weight
components is important because these may interfere with binding to the
purification columns. For example, ion-exchange chromatography depends on
ionic interactions between charged groups on the protein and oppositely
charged groups on the chromatography media. The high conductivity of the
harvest media would prevent protein from binding to the ion-exchange chro-
matography media.
Concentration and diafiltration are done using ultra-filtration membranes.
This operation allows for the passage of low-molecular-weight solutes and
water through membrane pores and the retention of larger-molecular-weight
solutes, like rHuEPO and other proteins. The driving force for the passage of
these solutes and water through the pores is the pressure difference across the
membrane. The membranes can be made of cellulose, polyether sulfone, or
other polymers. Membranes are available that have different “nominal mole-
cular-weight cut-offs,” ranging in molecular weight from 5,000 to 500,000.
Proteins and other high-molecular-weight materials that do not pass through
the membranes are referred to as the retentate; the material that passes through
the membranes is called the permeate. The rate at which the water and solutes
go through the membrane is called the permeate flux rate and is usually meas-
ured in liters per minute.
During the concentration phase (Fig. 10a), both water and solutes are forced
through the pores, leading to a volume reduction in the harvest medium. The
range of this volume reduction may be 5- to 100-fold depending on what is
required for a given protein. During the diafiltration phase, the concentrated
harvest continues to be fed through the membrane; at the same time, however,
a lower conductivity buffer (diafiltration buffer) is pumped into the harvest
