- Email: [email protected]
Biopharmaceutical technology: Cell harvesting — getting cultural
Feature
Filtration+Separation October 2008
Biopharmaceutical technology:
Cell harvesting – getting cultural P
eter Rose of Alfa Laval takes a look at one of the growth areas of biopharmaceutical filtration and separation: the harvesting of mammalian cells to produce new drugs and medicines. The article examines the origines of the technology used in this sector of the industry, and considers recent developments.
We – mammals that is – are composed of cells. The reflection that faces you in a mirror may look solid enough but what you are really seeing are approximately 10 trillion cells split into about 200 different types. These cell groups are quite specialised: livers are made of liver cells, muscles of specialist muscle cells and so on. Growing and harvesting mammalian cells outside of the body and in the laboratory or factory to produce new drugs and medicines is one of the most exciting arms of the modern life science industry. Yet, far-fetched as it may seem, these enormous advances in medical science can be traced back to a chicken which was dissected some time late in the 19th Century; 1885 to be precise. That was when Wilhelm Roux managed to maintain the medullary plate of the said embryonic chicken in a warm saline solution for a number of days and thus first established the principle of tissue culture.
filtration and filtration through absolute pore size membranes. Of the different techniques, centrifugation is the one most commonly scaled up from laboratory to factory production levels. Centrifugation uses the density difference between solids and the surrounding fluid and accelerates the settling that would normally occur during sedimentation. Most industrial applications use disk stack centrifuges to remove cells and cell debris from the nutrient broth. Disk stack centrifuges offer continuous operation, making their throughput consistent with the desire to limit the time for harvest operations.
His work was taken on several stages further by scientists at John Hopkins Medical School and Yale University during the first decade of the 20th Century. In the 1940s and 1950s viruses grown in cell cultures were used in the manufacture of vaccines. The polio vaccines that finally eliminated the threat of the disease were grown in cell cultures from monkey kidneys. These days, of course, drug production based on cell culture is one of the fastest-growing areas of biotechnology. Its rapid success owes much to the parallel development of technologies for the three key stages in cell culture growth: fermentation, harvesting and purification. Harvesting is performed by separating the cell culture from the growing medium and several techniques are used to perform this delicate operation; centrifugation, microfiltration, depth
Alfa Laval’s Culterfuge 100 cell separation system.
Naturally, it is not quite as simple as it appears. Mammalian cells are very fragile organisms so, although a disc stack centrifuge makes the job of separation relatively easy, the trick is to do so with minimal damage to the product. Acceleration of the protein rich feed material takes a fraction of a second. But although speed is of the essence, it must not be at expense of destroying the highly shearsensitive cell wall membrane which would release undesirable, intracellular proteins into the broth – a process known as lysis.
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Feature
Filtration+Separation October 2008
By preventing additional lysis during acceleration it is possible to increase the separator’s capacity while still achieving the required separation result. Downstream purification of target proteins is also simplified and can be carried out using more compact equipment, thus generating significant savings in the process. The challenge, then, is to achieve maximum separation efficiency with minimal product disruption. Just as cell culture has developed from relatively humble origins so has the equipment used to harvest it. In fact, from the earliest stages of cell culture science when Alfa Laval worked with industry leaders in the development of large scale cell culture fermentations, it soon became obvious that cell culture characteristics called for extremely gentle separator designs. What they turned to was the hollow spindle which owed its origins to concepts originally developed for the dairy industry where the gentle touch was used to prevent fat particles in milk from shearing apart during acceleration. Decades later, the same technology is a corner stone in modern cell culture processing. Like many innovations, the initial impetus had little to do with the eventual outcome. When Alfa Laval engineers originally decided to study the feasibility of a separator which provided liquid discharge under pressure, it was beer, not milk, which they had in mind. The target was to purify beer by separating it without air ingress and under sufficient pressure to prevent the loss of carbon dioxide. The only rational way in which to achieve a closed system was to feed the liquid to the bowl from underneath, through a tubular bowl spindle, and discharge it at the bowl neck. An experimental machine for clarifying beer was built but also tested on milk as it was felt that the machine might solve the milk froth problem: Which it did. The milk process then seemed to be the more commercially viable. The engineers modified the original machine to provide two outlets so that it could also be used as a cream separator. Trials in a Swedish dairy proved so successful
During the development of large scale cell culture fermentations, it soon became obvious that cell culture characteristics called for extremely gentle separator designs
that Alfa Laval then produced a number of the new separators for display at the 1933 Deutche Landwirt-schaftlische Gesellschaft exhibition in Berlin. This first hermetic separator radically changed dairy and brewery technology from that point onwards. As befitted a radically different piece of equipment, the hollow spindle hermetic separator looked totally different to earlier centrifuges. The frame was designed so that the inlets for whole milk at the bottom of the machine were readily accessible. Neither the traditional two-piece bowl spindle nor the newer Baltic type spindle could be used. Consequently, the hermetic separator had to incorporate an entirely new spindle design. The solution was to adapt the spindle from the largest Alfa Laval yeast separator at the time, the OVK 5. At the top end of the spindle, just beneath the bowl, the flexible top bearing – a radial ball bearing – supported the weight of the bowl, and at the bottom end, a spherical ball bearing allowed for the precision movement of the spindle. Whole milk was fed under pressure through the spindle into the bottom of the bowl. The seal between the stationary inlet pipe and the rotating spindle consisted of a U-shaped rubber gasket reinforced with fabric. Milk entered the bowl via the central chamber at its bottom, from where it was distributed in the disc stack in the usual way.
The Culterfuge cell harvesting module.
Since it did not have a hole through its centre its diameter was considerably reduced when compared to machines with top feed. As a result
of the overall design, the entire free space in the bowl was filled with liquid. Besides achieving the main objective – cream and skim milk free from foam – the hermetic separator provided several other advantages; the most important of which was its clean skimming capability which was better than any other separator. Two factors contributed to this improved level of performance. Firstly, the milk was accelerated very gently in the long hollow spindle, minimising the splitting of fat globules. Secondly, the Alfa-discs were given a smaller inner radius, increasing the separation area available in a given bowl volume. The same hollow spindle hermetic design is central to Culturefuge which was the world’s first fully hermetic purpose-built cell culture centrifuge, developed principally for applications involving mammalian cell cultures and precipitated protein. Its use was intended to provide the gentlest acceleration possible in a centrifugal disc stack separator designed specifically for cell culture harvesting. The smooth acceleration imposes minimal shear strains on the cells. As importantly, the hollow spindle creates a truly hermetic design that completely eliminates air-liquid interfaces within the centrifuge and thus also eliminates the foaming, a major cause of protein degradation. As a bonus, the design also allows centrate discharge at small radius which reduces both power consumption and keeps temperature pick up to a minimum during separation.
Feature
Filtration+Separation October 2008
In a mathematical model for feed zone breakage of shear sensitive particles in centrifugal separators, the breakage was found to be independent of flow-rate. A study by Boychyn et al. simulated the flow-field in the acceleration zone of a traditional centrifuge, using computational fluid dynamics (CFD). In this study two cases were compared, one classic with air present in the feed zone, and one gently liquid filled, i.e. with no air present.
identical. The group found a hollow spindle type centrifuge provided a 2.5-fold increase in throughput for the same clarification performance when compared to the other separator.
The modern Culturefuge system
Higher energy dissipation leads to a considerably higher particle breakage in the protein precipitate suspension and consequently less successful clarification. A later study by Boychyn, used a similar CFD tool to model the acceleration forces in the acceleration zone of a multi-chamber bowl. In this study, the group was able to make accurate predictions of centrifuge performance.
Translated into a modern cell culture harvesting system such as the Culturefuge, this hollow spindle technology made it possible to provide a method of continuous cell harvesting, under hermetic conditions. The skid-mounted Culturefuge consists of a diskstack, high-speed separator with piping for service liquids and process liquids. It includes an integrated electrical system with starter, PLC control and pneumatic unit. A motor with an integrated VFD is standard. As an option the system can also include a steamsterilisable pump for transport of the solids phase. Culturefuge’s design is in accordance with most major Pressure Vessel specifications including ASME and PED.
Finally, there is a comparative study by the same research group on Cell Culture processed in two small production/pilot scale disc stack centrifuges. One separator had a classic nonfilled acceleration zone and the other a hollow spindle hermetic inlet for gently accelerating the feed. In every other important aspect (equivalent area, rpm etc), the machines were
All product contact parts are made of highgrade stainless steel. Various grades of surface finish are available 1.2 µm Ra, 0.8 µm Ra or 0.5 µm Ra with electro-polish. The skidmounted modular design can be delivered for open operation, contained running, steam sterilisable aseptic operation or steam decontamination-only operation.
This modelling technique confirmed that, during acceleration with air present, the maximum energy dissipation rate is up to two times higher than in air free acceleration.
How it works Feed material enters the Culturefuge 100 through a hollow spindle feed inlet and accelerates gradually as it moves upwards, thereby minimising the shear forces on the liquids and preventing cell lysis. To prevent the risk of it mixing with air, the feed zone is completely filled with rotating liquid. The provision of this completely hermetic outlet eliminates the possibility of materials coming into contact with the air or the external environment; thus avoiding foaming and denaturisation of the product. During normal production the operating water keeps the sliding bowl bottom closed against the bowl hood. During discharge the sliding bowl bottom drops for a short time (less than a second) and the solids are ejected through the discharge ports. The high velocity of the ejected solids is reduced in the cyclone. Harvesting cells to develop new vaccines and medicines has moved from the realm of science fiction to fact, partly thanks to a slice of chicken and a separator that started life skimming milk from cream, and now helps harvest the most precious commodity of all, good health.
•
Contact: Peter Rose, Alfa Laval Email: [email protected] www.alfalaval.com
31
Filtration+Separation October 2008
Biopharmaceutical technology:
Cell harvesting – getting cultural P
eter Rose of Alfa Laval takes a look at one of the growth areas of biopharmaceutical filtration and separation: the harvesting of mammalian cells to produce new drugs and medicines. The article examines the origines of the technology used in this sector of the industry, and considers recent developments.
We – mammals that is – are composed of cells. The reflection that faces you in a mirror may look solid enough but what you are really seeing are approximately 10 trillion cells split into about 200 different types. These cell groups are quite specialised: livers are made of liver cells, muscles of specialist muscle cells and so on. Growing and harvesting mammalian cells outside of the body and in the laboratory or factory to produce new drugs and medicines is one of the most exciting arms of the modern life science industry. Yet, far-fetched as it may seem, these enormous advances in medical science can be traced back to a chicken which was dissected some time late in the 19th Century; 1885 to be precise. That was when Wilhelm Roux managed to maintain the medullary plate of the said embryonic chicken in a warm saline solution for a number of days and thus first established the principle of tissue culture.
filtration and filtration through absolute pore size membranes. Of the different techniques, centrifugation is the one most commonly scaled up from laboratory to factory production levels. Centrifugation uses the density difference between solids and the surrounding fluid and accelerates the settling that would normally occur during sedimentation. Most industrial applications use disk stack centrifuges to remove cells and cell debris from the nutrient broth. Disk stack centrifuges offer continuous operation, making their throughput consistent with the desire to limit the time for harvest operations.
His work was taken on several stages further by scientists at John Hopkins Medical School and Yale University during the first decade of the 20th Century. In the 1940s and 1950s viruses grown in cell cultures were used in the manufacture of vaccines. The polio vaccines that finally eliminated the threat of the disease were grown in cell cultures from monkey kidneys. These days, of course, drug production based on cell culture is one of the fastest-growing areas of biotechnology. Its rapid success owes much to the parallel development of technologies for the three key stages in cell culture growth: fermentation, harvesting and purification. Harvesting is performed by separating the cell culture from the growing medium and several techniques are used to perform this delicate operation; centrifugation, microfiltration, depth
Alfa Laval’s Culterfuge 100 cell separation system.
Naturally, it is not quite as simple as it appears. Mammalian cells are very fragile organisms so, although a disc stack centrifuge makes the job of separation relatively easy, the trick is to do so with minimal damage to the product. Acceleration of the protein rich feed material takes a fraction of a second. But although speed is of the essence, it must not be at expense of destroying the highly shearsensitive cell wall membrane which would release undesirable, intracellular proteins into the broth – a process known as lysis.
29
30
Feature
Filtration+Separation October 2008
By preventing additional lysis during acceleration it is possible to increase the separator’s capacity while still achieving the required separation result. Downstream purification of target proteins is also simplified and can be carried out using more compact equipment, thus generating significant savings in the process. The challenge, then, is to achieve maximum separation efficiency with minimal product disruption. Just as cell culture has developed from relatively humble origins so has the equipment used to harvest it. In fact, from the earliest stages of cell culture science when Alfa Laval worked with industry leaders in the development of large scale cell culture fermentations, it soon became obvious that cell culture characteristics called for extremely gentle separator designs. What they turned to was the hollow spindle which owed its origins to concepts originally developed for the dairy industry where the gentle touch was used to prevent fat particles in milk from shearing apart during acceleration. Decades later, the same technology is a corner stone in modern cell culture processing. Like many innovations, the initial impetus had little to do with the eventual outcome. When Alfa Laval engineers originally decided to study the feasibility of a separator which provided liquid discharge under pressure, it was beer, not milk, which they had in mind. The target was to purify beer by separating it without air ingress and under sufficient pressure to prevent the loss of carbon dioxide. The only rational way in which to achieve a closed system was to feed the liquid to the bowl from underneath, through a tubular bowl spindle, and discharge it at the bowl neck. An experimental machine for clarifying beer was built but also tested on milk as it was felt that the machine might solve the milk froth problem: Which it did. The milk process then seemed to be the more commercially viable. The engineers modified the original machine to provide two outlets so that it could also be used as a cream separator. Trials in a Swedish dairy proved so successful
During the development of large scale cell culture fermentations, it soon became obvious that cell culture characteristics called for extremely gentle separator designs
that Alfa Laval then produced a number of the new separators for display at the 1933 Deutche Landwirt-schaftlische Gesellschaft exhibition in Berlin. This first hermetic separator radically changed dairy and brewery technology from that point onwards. As befitted a radically different piece of equipment, the hollow spindle hermetic separator looked totally different to earlier centrifuges. The frame was designed so that the inlets for whole milk at the bottom of the machine were readily accessible. Neither the traditional two-piece bowl spindle nor the newer Baltic type spindle could be used. Consequently, the hermetic separator had to incorporate an entirely new spindle design. The solution was to adapt the spindle from the largest Alfa Laval yeast separator at the time, the OVK 5. At the top end of the spindle, just beneath the bowl, the flexible top bearing – a radial ball bearing – supported the weight of the bowl, and at the bottom end, a spherical ball bearing allowed for the precision movement of the spindle. Whole milk was fed under pressure through the spindle into the bottom of the bowl. The seal between the stationary inlet pipe and the rotating spindle consisted of a U-shaped rubber gasket reinforced with fabric. Milk entered the bowl via the central chamber at its bottom, from where it was distributed in the disc stack in the usual way.
The Culterfuge cell harvesting module.
Since it did not have a hole through its centre its diameter was considerably reduced when compared to machines with top feed. As a result
of the overall design, the entire free space in the bowl was filled with liquid. Besides achieving the main objective – cream and skim milk free from foam – the hermetic separator provided several other advantages; the most important of which was its clean skimming capability which was better than any other separator. Two factors contributed to this improved level of performance. Firstly, the milk was accelerated very gently in the long hollow spindle, minimising the splitting of fat globules. Secondly, the Alfa-discs were given a smaller inner radius, increasing the separation area available in a given bowl volume. The same hollow spindle hermetic design is central to Culturefuge which was the world’s first fully hermetic purpose-built cell culture centrifuge, developed principally for applications involving mammalian cell cultures and precipitated protein. Its use was intended to provide the gentlest acceleration possible in a centrifugal disc stack separator designed specifically for cell culture harvesting. The smooth acceleration imposes minimal shear strains on the cells. As importantly, the hollow spindle creates a truly hermetic design that completely eliminates air-liquid interfaces within the centrifuge and thus also eliminates the foaming, a major cause of protein degradation. As a bonus, the design also allows centrate discharge at small radius which reduces both power consumption and keeps temperature pick up to a minimum during separation.
Feature
Filtration+Separation October 2008
In a mathematical model for feed zone breakage of shear sensitive particles in centrifugal separators, the breakage was found to be independent of flow-rate. A study by Boychyn et al. simulated the flow-field in the acceleration zone of a traditional centrifuge, using computational fluid dynamics (CFD). In this study two cases were compared, one classic with air present in the feed zone, and one gently liquid filled, i.e. with no air present.
identical. The group found a hollow spindle type centrifuge provided a 2.5-fold increase in throughput for the same clarification performance when compared to the other separator.
The modern Culturefuge system
Higher energy dissipation leads to a considerably higher particle breakage in the protein precipitate suspension and consequently less successful clarification. A later study by Boychyn, used a similar CFD tool to model the acceleration forces in the acceleration zone of a multi-chamber bowl. In this study, the group was able to make accurate predictions of centrifuge performance.
Translated into a modern cell culture harvesting system such as the Culturefuge, this hollow spindle technology made it possible to provide a method of continuous cell harvesting, under hermetic conditions. The skid-mounted Culturefuge consists of a diskstack, high-speed separator with piping for service liquids and process liquids. It includes an integrated electrical system with starter, PLC control and pneumatic unit. A motor with an integrated VFD is standard. As an option the system can also include a steamsterilisable pump for transport of the solids phase. Culturefuge’s design is in accordance with most major Pressure Vessel specifications including ASME and PED.
Finally, there is a comparative study by the same research group on Cell Culture processed in two small production/pilot scale disc stack centrifuges. One separator had a classic nonfilled acceleration zone and the other a hollow spindle hermetic inlet for gently accelerating the feed. In every other important aspect (equivalent area, rpm etc), the machines were
All product contact parts are made of highgrade stainless steel. Various grades of surface finish are available 1.2 µm Ra, 0.8 µm Ra or 0.5 µm Ra with electro-polish. The skidmounted modular design can be delivered for open operation, contained running, steam sterilisable aseptic operation or steam decontamination-only operation.
This modelling technique confirmed that, during acceleration with air present, the maximum energy dissipation rate is up to two times higher than in air free acceleration.
How it works Feed material enters the Culturefuge 100 through a hollow spindle feed inlet and accelerates gradually as it moves upwards, thereby minimising the shear forces on the liquids and preventing cell lysis. To prevent the risk of it mixing with air, the feed zone is completely filled with rotating liquid. The provision of this completely hermetic outlet eliminates the possibility of materials coming into contact with the air or the external environment; thus avoiding foaming and denaturisation of the product. During normal production the operating water keeps the sliding bowl bottom closed against the bowl hood. During discharge the sliding bowl bottom drops for a short time (less than a second) and the solids are ejected through the discharge ports. The high velocity of the ejected solids is reduced in the cyclone. Harvesting cells to develop new vaccines and medicines has moved from the realm of science fiction to fact, partly thanks to a slice of chicken and a separator that started life skimming milk from cream, and now helps harvest the most precious commodity of all, good health.
•
Contact: Peter Rose, Alfa Laval Email: [email protected] www.alfalaval.com
31