The Manufacturing of the Recombinant Factor VIII, Kogenate Berthold G.D. Boedeker
R
ECOMBINANT factor VIII (rFVIII) (Kogenate; Miles, Inc, Berkeley, CA) for the treatment of hemophilia A is produced from mammalian cells (baby hamster kidney [BHK]) transfected with the human FVIII gene. These cells are cultured in a specifically designed, bovine serum-free medium that allows high-yield rFVIII expression without the addition of von Willebrand factor for stabilization of the FVIII molecule. Large-scale fermentation is performed in deep-tank fermenters using a continuous, high cell density cultivation process. The fermenter cultures are operated in a perfusion mode for up to 6 months under steady state conditions, allowing a higher degree of process control as compared with batch or repeated fed-batch production. The rFVIII raw product from fermentation is processed batch-wise through a multi-step purification system, resulting in a final product of high purity and specific activity. A series of purification steps, including ion exchange, size exclusion, and immunoaffinity chromatographies, are used to remove effectively contaminants such as culture medium components, host cell DNA, and host cell proteins, as well as impurities derived from the purification process itself, such as murine immunoglobulin G (lgG), from the immunoaffinity chromatography step. In addition, the purification process has the capability to inactivate and remove viruses achieved by a virus inactivation step and by virus clearance through the individual purification steps. Finally, the purified rFVIII is formulated, filled, and freeze-dried (Fig l). Product quality is assured by an extensive quality assurance program, including controls of facilities, utilities and equipment, raw materials, inprocess and final product testing as well as the validation of the fermentation and purification
From the Department oj Celi Culture Technology and Production, Miles Inc., Berkeley. CA. This article is a summary oj the presentation given at the International Symposium on Recombinant Factor VIII. Address reprint requests to Berthold G.D. Boedeker, PhD, Director, Celi Culture Technology and Production, Mi/es Inc., 4th and Parker Sts, Berkeley, CA 94701. Copyright © 1992 by W.B. Saunders Company 0887-7963/92/0604-0004$3.00/0
256
steps. The manufacturing process was successfully scaled-up and is routinely run in a large-scale production facility located in Berkeley, CA. The development of an efficient large-scale manufacturing process for rFVIII from mammalian cell cultures is difficult and time consuming because the cells release this very large glycoprotein only in small quantities into the medium. In addition, the FVIII molecule itself is not very stable even in its naturai environment, the human body. The major development goals that have to be achieved for production of a recombinant protein from mammalian cells are summarized in Fig 2. The first step is to optimize the expression system, the producing cell line. After that, a cell bank has to be prepared to assure that each production campaign starts from the identical source of cells. This is achieved by freezing small quantities of cells in 1- to 2-mL containers and storing them in liquid nitrogen. The next step is the development of an appropriate culture medium that supports cell growth as well as product formation and product stability . Therefore, a technical culture system has to be developed that allows the product to be produced reproducibly under controlled culture conditions. In addition, this system has to be adaptable to the manufacturing scale. In parallei, a purification process must be developed to allow effective removal of impurities such as components used in the manufacturing process as well as host cell DNA and host cell proteins. Finally, there are two scale-up steps involved in the development cascade: (1) the pilot plant, and (2) the production plant. In the following sections, fermentation and purification of rFVIII from BHK cells transfected with the human rFVIII gene and the production plant will be described in more detail. CELL CULTIVATION-RECOMBINANT FACTOR VIII FERMENTATION
All cell cultivation, starting from the expansion of the cell bank to the final scale production in fermenters, is performed in a specifically designed culture medium that contains the three protein Transfusion Medicine Reviews, Vol VI, No 4 IOctober), 1992: pp 256-260
257
MANUFACTURING OF KOGENATE
r - - - - Dxygen r - - - - Carbohydrates
Proteins rFVI//
DNA
BHK Ce//s
Harvest
Chromatographlc Steps
B/oreactor (Fermenter) Excip/(Jflls
(e.g.A/bumlnr-I}
..J
~. Formulat/on
Reconstltute w//h
~/water
~-t-J FreezeDry/ilg
Sten/eFI/trat/on Fig 1.
Flila/ Product
'/--1\•
Synilge
Patlent
The manufacturing process of rFVIII.
compounds of transferrin, insulin, and human serum albumin. In addition, the medium cantains all nutrients, vitamins, hormones, and salts that are needed by mammalian celis for growth and metabolic activity. The rFVIII medium supports celi growth, gives high rFVIII yields, and stabilizes the FVIII molecule. It is completely bovine serum-free and does not contain any von Willebrand factor for stabilization af the molecule. The validated technical culture system used for rFVIII production is based on a fermentation system well known for microbial fermentation. The cells are cultivated in suspension culture in agitated deep-tank fermenters. The fermenters are operated for up to 6 months under sterile conditians. During this time, culture fluid containing rFVIII is removed continuously from the fermenter at the same rate as fresh culture fluid is added back (Fig 3). Thus, the cultures are operated under steady state physiological conditions allowing a high degree of process control. To avoid excessive loss of celis during harvest, they are separated from the harvest fluid and re-
Culture Medium Development
Development ot a Technical Culture System
Development ot a Purification Process
/ Scale-up Step 1: Pilot Plant
Scale-up Step 2: Production Plant Fig 2. The production of recombinant products from mam· malian celi culture.
BERTHOLD G.D. BOEDEKER
258
Celi Retention System
l
Fermenter
Feed Medium Tank
tained in the fermenter. Such retention of cells may be achieved by rotating spin filters, continuous centrifugation, sedimentation devices, or rotating microfiltration devices. Indeed, using in-housedeveloped retention systems, the rFVIII cultures contain up to 30-fold higher cell concentrations than usually possible without cell retention. This is particularly important because mammalian cells grow very slowly compared with bacteria and other microorganisms, with doubling times of l to 3 days. The successful retention of cells in the fermenter therefore makes the cultivation process highly efficient. To reproducibly produce rFVIII under controlled culture conditions, a variety of culture parameters are monitored and controlled (Table l). First, there are the basic fermentation parameters such as oxygen concentration, pH, temperature, and agitation speed, which are continuously monitored on-line. Other parameters, such as glucose concentration (as an indicator for the nutrient state of the medium), rFVIll concentration, cell density and cell viability, perfusion rate, sterility, and absence of mycoplasma and viruses, are monitored off-line daily or in larger intervals from fermenter samples. In addition to these directly measured parameters, there are calculated parameters, such as specific rFVIII production, specific glucose consumption, and specific perfusion rate, which indi-
Harvest Tank
Fig 3. lhe rFVIII fermentation process.
cate how the cells perform during the cultivation period. The continuous perfusion culture at high cell density used for rFVIll production provides two major advantages compared with standard batch or repeated batch cultures that are most frequently used for mammalian cell fermentation. The first advantage is a high degree of culture controI. In a perfusion culture the optimal conditions regarding
labie 1. rFVIII Fermentatlon Controlled and Monitored Culture Parameters Measured parameters Oxygen concentration pH
Temperature Agitatlon speed Glucose concentration Absence of mycoplasmas and viruses rFVlI1 concentration Perfusion rate Celi density Celi viability Sterility Calculated parameters Specific rFVl1I production (per cell/day) Specific glucose consumption Specific perfusion rate Volumetric rFV1I1 production (per liter fermenter volume/day)
MANUFACTURING OF KOGENATE
medium components can be adjusted and kept constant. This results in real steady state physiological conditions during fermentation. In batch or repeated batch cultures on the other hand, there are gradients regarding medium nutrients during each cultivation cycle, ranging from optimal conditions in the beginning of a cycle after fresh medium has been added, to poor conditions at the end of a cycle when most of the nutrients have already been consumed. The second advantage of the high cell density perfusion culture is a large reduction in the necessary fermentation volume. Because the specific rFVIII production, ie, the production per cell and day, is not affected by the actual cell density , a perfusion fermenter with a 30-fold increased cell density produces 30-fold more rFVIII than cultures without cell retention. This means that a 100-L perfusion culture produces as much as a 3,OOO-L batch or repeated batch culture. Therefore, the largest high cell density fermenter that is needed for rFVIII production is only 500 L, which is much easier to operate and control than a corresponding 15,000-L fermenter for low cell density cultures. RECOMBINANT FACTOR VIII PURIFICATION
The rFVIII raw product from fermentation is processed batch-wise through a validated multistep purification system resulting in a final product with high purity and specific activity (Table 2). In addition, the purification process has the capability
Table 2. Schematic rFVlI1 Purification Process Fermenter harvest
1 Celi separation
1 Anion-exchange chromatography
1 Virus inactivation
1 Immunoadsorption chromatography
1 Size-exclusion chromatography
J, Anion-exchange chromatography
J, Buffer-exchange formulation
1 Sterile filtration/lyophilization
259
to remove and inactivate viruses, which is a regulatory requirement, ałthough the cells are virusfree. In our process, this requirement is achieved by a virus inactivation step and by virus clearance through the individual purification steps. Using a broad spectrum of viruses with very different physico-chemicał characteristics, the purification process was shown to be capable of removing viruses with titer reductions of at least 106 to more than 10 12 . The first step in the purification process is celI separation, in which remaining cells are removed from the harvest. This is necessary because most of the cell retention systems available do not retain celIs to 100% in the fermenter. The purification process itself is a multi-step process in which the primary principles of separation of biologics, ie, charge, size, and affinity are used to maximize removal of impurities. It starts with an anionexchange chromatography to concentrate the product and to remove the majority of the culture medium proteins as well as some host cell DNA. The key purification step is immunoadsorption chromatography using monoclonal antibodies against FVIII. The antibodies are produced from mammalian celI culture in a serum-free culture process, purified, and coupled to a gel matrix before they are used for rFVIII purification. The immunoadsorption chromatography removes the majority of host cell proteins as well as the remaining DNA contaminants. The following steps, size exclusion and another anion-exchange chromatography, are performed to remove the very small amount of antibodies leaching from the immunoadsorption column. The purified rFVIII is formulated by adjusting excipients and adding human serum albumin to stabilize the product. FinalIy, it is fi11ed and freeze-dried. Product quałity is assured by an extensive quality assurance program including raw materiaIs , in process and finał product testing, as well as controls of facilities, utilities, and equipment. RECOMBINANT FACTOR VIII PRODUG~']l'ION PLANT
The production process for rFVIII was successfully brought up to a manufacturing scałe of operation in a plant located in Berkeley, CA. The plant accommodates the complete manufacturing process from storage of the cell bank to preparation of
260
the final freeze-dried containers. It consists of five major departments: media for cell cultivation and fermentation as well as buffers for purification are prepared in the media prep area; there are two independent fermentation areas; harvests from both fermentation areas are processed in the purification area. The chromatography steps are performed in a cold room on individual purification skids.
BERTHOLD G.D. BOEDEKER
To operate the cultures continuously for up to 6 months there are. two medium hold tanks and two harvest tanks for each production fermenter. One of these tanks is always in use while the other is undergoing maintenance and sterility testing. Most systems in the plant are operated by a decentralized computer system. The facility is validated and in full operation.
R
ECOMBINANT factor VIII (rFVIII) (Kogenate; Miles, Inc, Berkeley, CA) for the treatment of hemophilia A is produced from mammalian cells (baby hamster kidney [BHK]) transfected with the human FVIII gene. These cells are cultured in a specifically designed, bovine serum-free medium that allows high-yield rFVIII expression without the addition of von Willebrand factor for stabilization of the FVIII molecule. Large-scale fermentation is performed in deep-tank fermenters using a continuous, high cell density cultivation process. The fermenter cultures are operated in a perfusion mode for up to 6 months under steady state conditions, allowing a higher degree of process control as compared with batch or repeated fed-batch production. The rFVIII raw product from fermentation is processed batch-wise through a multi-step purification system, resulting in a final product of high purity and specific activity. A series of purification steps, including ion exchange, size exclusion, and immunoaffinity chromatographies, are used to remove effectively contaminants such as culture medium components, host cell DNA, and host cell proteins, as well as impurities derived from the purification process itself, such as murine immunoglobulin G (lgG), from the immunoaffinity chromatography step. In addition, the purification process has the capability to inactivate and remove viruses achieved by a virus inactivation step and by virus clearance through the individual purification steps. Finally, the purified rFVIII is formulated, filled, and freeze-dried (Fig l). Product quality is assured by an extensive quality assurance program, including controls of facilities, utilities and equipment, raw materials, inprocess and final product testing as well as the validation of the fermentation and purification
From the Department oj Celi Culture Technology and Production, Miles Inc., Berkeley. CA. This article is a summary oj the presentation given at the International Symposium on Recombinant Factor VIII. Address reprint requests to Berthold G.D. Boedeker, PhD, Director, Celi Culture Technology and Production, Mi/es Inc., 4th and Parker Sts, Berkeley, CA 94701. Copyright © 1992 by W.B. Saunders Company 0887-7963/92/0604-0004$3.00/0
256
steps. The manufacturing process was successfully scaled-up and is routinely run in a large-scale production facility located in Berkeley, CA. The development of an efficient large-scale manufacturing process for rFVIII from mammalian cell cultures is difficult and time consuming because the cells release this very large glycoprotein only in small quantities into the medium. In addition, the FVIII molecule itself is not very stable even in its naturai environment, the human body. The major development goals that have to be achieved for production of a recombinant protein from mammalian cells are summarized in Fig 2. The first step is to optimize the expression system, the producing cell line. After that, a cell bank has to be prepared to assure that each production campaign starts from the identical source of cells. This is achieved by freezing small quantities of cells in 1- to 2-mL containers and storing them in liquid nitrogen. The next step is the development of an appropriate culture medium that supports cell growth as well as product formation and product stability . Therefore, a technical culture system has to be developed that allows the product to be produced reproducibly under controlled culture conditions. In addition, this system has to be adaptable to the manufacturing scale. In parallei, a purification process must be developed to allow effective removal of impurities such as components used in the manufacturing process as well as host cell DNA and host cell proteins. Finally, there are two scale-up steps involved in the development cascade: (1) the pilot plant, and (2) the production plant. In the following sections, fermentation and purification of rFVIII from BHK cells transfected with the human rFVIII gene and the production plant will be described in more detail. CELL CULTIVATION-RECOMBINANT FACTOR VIII FERMENTATION
All cell cultivation, starting from the expansion of the cell bank to the final scale production in fermenters, is performed in a specifically designed culture medium that contains the three protein Transfusion Medicine Reviews, Vol VI, No 4 IOctober), 1992: pp 256-260
257
MANUFACTURING OF KOGENATE
r - - - - Dxygen r - - - - Carbohydrates
Proteins rFVI//
DNA
BHK Ce//s
Harvest
Chromatographlc Steps
B/oreactor (Fermenter) Excip/(Jflls
(e.g.A/bumlnr-I}
..J
~. Formulat/on
Reconstltute w//h
~/water
~-t-J FreezeDry/ilg
Sten/eFI/trat/on Fig 1.
Flila/ Product
'/--1\•
Synilge
Patlent
The manufacturing process of rFVIII.
compounds of transferrin, insulin, and human serum albumin. In addition, the medium cantains all nutrients, vitamins, hormones, and salts that are needed by mammalian celis for growth and metabolic activity. The rFVIII medium supports celi growth, gives high rFVIII yields, and stabilizes the FVIII molecule. It is completely bovine serum-free and does not contain any von Willebrand factor for stabilization af the molecule. The validated technical culture system used for rFVIII production is based on a fermentation system well known for microbial fermentation. The cells are cultivated in suspension culture in agitated deep-tank fermenters. The fermenters are operated for up to 6 months under sterile conditians. During this time, culture fluid containing rFVIII is removed continuously from the fermenter at the same rate as fresh culture fluid is added back (Fig 3). Thus, the cultures are operated under steady state physiological conditions allowing a high degree of process control. To avoid excessive loss of celis during harvest, they are separated from the harvest fluid and re-
Culture Medium Development
Development ot a Technical Culture System
Development ot a Purification Process
/ Scale-up Step 1: Pilot Plant
Scale-up Step 2: Production Plant Fig 2. The production of recombinant products from mam· malian celi culture.
BERTHOLD G.D. BOEDEKER
258
Celi Retention System
l
Fermenter
Feed Medium Tank
tained in the fermenter. Such retention of cells may be achieved by rotating spin filters, continuous centrifugation, sedimentation devices, or rotating microfiltration devices. Indeed, using in-housedeveloped retention systems, the rFVIII cultures contain up to 30-fold higher cell concentrations than usually possible without cell retention. This is particularly important because mammalian cells grow very slowly compared with bacteria and other microorganisms, with doubling times of l to 3 days. The successful retention of cells in the fermenter therefore makes the cultivation process highly efficient. To reproducibly produce rFVIII under controlled culture conditions, a variety of culture parameters are monitored and controlled (Table l). First, there are the basic fermentation parameters such as oxygen concentration, pH, temperature, and agitation speed, which are continuously monitored on-line. Other parameters, such as glucose concentration (as an indicator for the nutrient state of the medium), rFVIll concentration, cell density and cell viability, perfusion rate, sterility, and absence of mycoplasma and viruses, are monitored off-line daily or in larger intervals from fermenter samples. In addition to these directly measured parameters, there are calculated parameters, such as specific rFVIII production, specific glucose consumption, and specific perfusion rate, which indi-
Harvest Tank
Fig 3. lhe rFVIII fermentation process.
cate how the cells perform during the cultivation period. The continuous perfusion culture at high cell density used for rFVIll production provides two major advantages compared with standard batch or repeated batch cultures that are most frequently used for mammalian cell fermentation. The first advantage is a high degree of culture controI. In a perfusion culture the optimal conditions regarding
labie 1. rFVIII Fermentatlon Controlled and Monitored Culture Parameters Measured parameters Oxygen concentration pH
Temperature Agitatlon speed Glucose concentration Absence of mycoplasmas and viruses rFVlI1 concentration Perfusion rate Celi density Celi viability Sterility Calculated parameters Specific rFVl1I production (per cell/day) Specific glucose consumption Specific perfusion rate Volumetric rFV1I1 production (per liter fermenter volume/day)
MANUFACTURING OF KOGENATE
medium components can be adjusted and kept constant. This results in real steady state physiological conditions during fermentation. In batch or repeated batch cultures on the other hand, there are gradients regarding medium nutrients during each cultivation cycle, ranging from optimal conditions in the beginning of a cycle after fresh medium has been added, to poor conditions at the end of a cycle when most of the nutrients have already been consumed. The second advantage of the high cell density perfusion culture is a large reduction in the necessary fermentation volume. Because the specific rFVIII production, ie, the production per cell and day, is not affected by the actual cell density , a perfusion fermenter with a 30-fold increased cell density produces 30-fold more rFVIII than cultures without cell retention. This means that a 100-L perfusion culture produces as much as a 3,OOO-L batch or repeated batch culture. Therefore, the largest high cell density fermenter that is needed for rFVIII production is only 500 L, which is much easier to operate and control than a corresponding 15,000-L fermenter for low cell density cultures. RECOMBINANT FACTOR VIII PURIFICATION
The rFVIII raw product from fermentation is processed batch-wise through a validated multistep purification system resulting in a final product with high purity and specific activity (Table 2). In addition, the purification process has the capability
Table 2. Schematic rFVlI1 Purification Process Fermenter harvest
1 Celi separation
1 Anion-exchange chromatography
1 Virus inactivation
1 Immunoadsorption chromatography
1 Size-exclusion chromatography
J, Anion-exchange chromatography
J, Buffer-exchange formulation
1 Sterile filtration/lyophilization
259
to remove and inactivate viruses, which is a regulatory requirement, ałthough the cells are virusfree. In our process, this requirement is achieved by a virus inactivation step and by virus clearance through the individual purification steps. Using a broad spectrum of viruses with very different physico-chemicał characteristics, the purification process was shown to be capable of removing viruses with titer reductions of at least 106 to more than 10 12 . The first step in the purification process is celI separation, in which remaining cells are removed from the harvest. This is necessary because most of the cell retention systems available do not retain celIs to 100% in the fermenter. The purification process itself is a multi-step process in which the primary principles of separation of biologics, ie, charge, size, and affinity are used to maximize removal of impurities. It starts with an anionexchange chromatography to concentrate the product and to remove the majority of the culture medium proteins as well as some host cell DNA. The key purification step is immunoadsorption chromatography using monoclonal antibodies against FVIII. The antibodies are produced from mammalian celI culture in a serum-free culture process, purified, and coupled to a gel matrix before they are used for rFVIII purification. The immunoadsorption chromatography removes the majority of host cell proteins as well as the remaining DNA contaminants. The following steps, size exclusion and another anion-exchange chromatography, are performed to remove the very small amount of antibodies leaching from the immunoadsorption column. The purified rFVIII is formulated by adjusting excipients and adding human serum albumin to stabilize the product. FinalIy, it is fi11ed and freeze-dried. Product quałity is assured by an extensive quality assurance program including raw materiaIs , in process and finał product testing, as well as controls of facilities, utilities, and equipment. RECOMBINANT FACTOR VIII PRODUG~']l'ION PLANT
The production process for rFVIII was successfully brought up to a manufacturing scałe of operation in a plant located in Berkeley, CA. The plant accommodates the complete manufacturing process from storage of the cell bank to preparation of
260
the final freeze-dried containers. It consists of five major departments: media for cell cultivation and fermentation as well as buffers for purification are prepared in the media prep area; there are two independent fermentation areas; harvests from both fermentation areas are processed in the purification area. The chromatography steps are performed in a cold room on individual purification skids.
BERTHOLD G.D. BOEDEKER
To operate the cultures continuously for up to 6 months there are. two medium hold tanks and two harvest tanks for each production fermenter. One of these tanks is always in use while the other is undergoing maintenance and sterility testing. Most systems in the plant are operated by a decentralized computer system. The facility is validated and in full operation.
