Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/06—Lysis of microorganisms
  • C12N1/066—Lysis of microorganisms by physical methods
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
  • C12N2710/16751—Methods of production or purification of viral material

Definitions

• This invention relates to a method for the disruption of cells grown in culture by using opposing jet streams operating at a low pressure to create a disruptive fluid shear which is powerful enough to disrupt the cells, but not so powerful as to destroy their contents.
• Rotor/stator devices having a cylindrical rotor turning at high speed concentrically inside a stator may be used to disrupt animal cells. These devices create a steep velocity gradient in the annular region generating sufficient shear stress in the fluid to disrupt the cells.
• a similar device called the Chaikoff Press has a cylinder and a piston of a slightly smaller diameter. Movement of the piston creates high shear in the fluid within the annular space, causing cell rupture.
• This type of device is also referred to as a "douncer" and has been used for the disruption of MRC-5 diploid lung cells infected with Varicella.
• these methods are only workable at the laboratory scale, and are not amenable to scale-up for manufacturing.
• Ultrasonics or sonication disrupts cells by creating high shear stress regions in the fluid through the process of cavitation.
• Oscillating acoustic waves ⁇ 20kHz
• Vapor bubbles formed in the low pressure region collapse upon entering the high pressure region causing high energy shock waves.
• shock wave moves radially from the initial cavitation site, high shear stresses are generated as well as heat as the energy dissipates in the fluid.
• a continuous flow sonication device has been used to disrupt MRC-5 diploid lung cells containing Varicella virus.
• the region in close proximity to the origin of the shock wave is of sufficient energy to destroy Varicella infectivity. Therefore, the amount of virus infectivity lost depends on the number of nucleation sites generated per unit volume fluid which is related to the energy input into the fluid.
• the continuous sonication process also results in a 5 to 10°C temperature rise in the process fluid, which increases the degradation rate of the virus.
• Sonication energy input can be lowered to increase post sonication titers; however, less disruption results in greater losses across the clarifying filters.
• An optimization exists between infectious titer loss due to sonication and the degree of cell disruption required for high titers post clarification.
• freeze/thaws are used to maximize the release of Rotavirus from culture. This method, while useful, is difficult to implement in manufacturing.
• Two impinging jet methodologies are known: impingement of a fluid jet on a plate and impingement on an opposing jet.
• the impinging region produces a micromixing zone where the shear is controlled by the linear velocity of the jet.
• These devices have been used for the disruption of microbial cells.
• the MICROFLUIDIZER device uses an interaction chamber where two jet streams impinge on one another at linear velocities up to 200+ m/s.
• the mode of disruption are reported as cavitation, fluid shear and impact.
• this device is not suitable for use with animal cells because not only are cells disrupted, but their contents are damaged as well. It would be desirable to have a device which efficiently disrupts animal cells and releases the cell content without damage.
• This invention relates to a novel method of disrupting cultured cells which lack a cell wall comprising passing cells suspended in a suspension fluid through a low pressure impinging jet device.
• Another aspect of this invention is a method of harvesting a cell product contained within a cell which does not have a cell wall comprising: a) culturing cells under culture conditions in a culture medium until the cell product is produced; b) suspending the cells in a suspension fluid; c) passing the suspended cells through a low pressure impinging jet device so that the cells are disrupted at a pressure of from about 5 to 100 psi and the cell product is released; and d) recovering the released cell product.
• the method of this invention may be broadly applied to any cell which lacks a cell wall or which has had its cell wall removed. While animal cells are preferred, this method works equally well with other cells, such as plant or fungal protoplasts and bacterial spheroplasts. The only requirement is that the cells be amenable to cell culture, and it is preferred that the cells be amenable to a large scale culture. Examples of suitable animal cells include VERO cells, CHO cells, and diploid fibroblast cells such as MRC-5 cells. Examples of suitable plant protoplasts include Nicotiana, Petunia, Zea, Brassica, and interspecial hybrids. The cells lines may be immortalized or not, and they may be cultivated in either a stationary or in a suspension culture. None of the particular culture parameters are critical to the method of this invention.
• the method of this invention may be used to recover virtually any type of product which is made using the cultured cells.
• examples of such products include: naturally occurring products, such as proteins; polysaccharides; recombinant proteins, including antibodies and enzymes; and viruses.
• animal cells are used as host cells for the production of viruses which are used in the manufacture of vaccines.
• this invention comprises method of harvesting a virus grown in an animal cell comprising: a) culturing animal cells infected with the virus; b) suspending the animal cells containing the virus in a suspension fluid; c) passing the suspended animal cells through a low pressure impinging jet device so that cells are disrupted and the virus is released; and d) harvesting the released virus.
• MRC-5 human diploid lung cells infected Varicella Zoster Virus are disrupted to harvest virus used to prepare a live virus vaccine, VARIVAX®.
• Figure 1 is a drawing of an impinging jet device which may be used in the method of this invention.
• the cells are cultured as is customary for the particular cell. After a suitable culture period, the cells are released from their substrate (if they are anchored), suspended in a fluid.
• the fluid may be the same or similar to that used to culture the cells, or it may be a stabilizer.
• the composition of the suspension fluid is not critical.
• the suspended cells are processed through an impinging jet device such as that shown in Figure 1.
• fluid shear is preferably generated by impinging two opposing jet streams 111 and 101 at a controlled linear velocity in a small chamber 120.
• the device is preferably operated in a continuous mode with the input stream split into a two jet streams 100, 110 and an outlet stream 130 draining the chamber. It is desirable that nozzles 111 and 101 be placed in close proximity to each other, i.e. less than one inch apart, and more preferably approximately l/8th to 3/8ths of an inch apart in order to maximize the fluid shear.
• a critical aspect of this method is that the device is operated at a low pressure, in a non-cavitating mode, preferably less than about 150 psi, and more preferably less than about 100 psi.
• the low operating pressure results in a gentle disruption— cells are preferably ruptured at a very low pressure, from about 5 to about 100 psi.
• a commercially available impinging jet cell disrupter sold under the tradename MICROFLUIDIZER® by Microfluidics International Corp., Newton, MA, reports disruption of animal cells at 2,000 psi. At this high pressure, cavitation and its damaging effects are likely to occur.
• the linear jet velocity at the point of impingement is also a critical aspect of this invention since it dictates the disruptive force.
• the linear jet velocity be approximately 5 to 50 m/s, and preferably 10 to 30 m/s.
• the method of this invention has been designed specifically for disruption of cells without cell walls, where a lower energy input is delivered in a controlled way. It is difficult for a high pressure device such as an homogenizer or MICROFLUIDIZER® which are designed to deliver up to 15,000 and 40,000 psi, respectively, to deliver precise control at low pressures well below their respective design specifications whereas the method of this invention preferably uses a device which optimally runs at a low pressure.
• the impinging jet process of this invention has been shown to provide adequate cell breakage for high filtration yields with negligible loss of infectious titer. In preliminary studies the impinging jet provided superior yields to the freeze/thaw approach.
• the method of this invention has a number of advantages over the current methods of cell disruption.
• the device is simple in design and does not require a piston pump nor cooling device, avoiding problems associated with these types of components.
• the low pressure operating system has the further advantage of conveniently allowing a low pressure processing pump (lobe, or diaphragm) or a relatively low pressure vessel to be used to drive the fluid.
• the impinging jet device disrupts the cells by fluid shear created by micromixing in a well defined impingement zone. Cavitation does not occur under the disruption conditions used. Heterogeneous zones of damaging high shear stress common to cavitation based disruption mechanisms are avoided.
• the impinging jet device is scaleable.
• the volumetric processing rate at a given linear velocity can be increased by increasing the jet orifice.
• the device is sanitary in design and uses commercially available nozzle technology for consistent fabrication, and can be inco ⁇ orated directly into standard process equipment. Further, the device can be sterilized in place.
• the impinging jet cell disruption method of this invention can be used for high yield recovery of Varicella Zoster Virus, other viruses or intracellular proteins from animal cells. After disruption, cell debris is separated from the associated virus particles by clarifying filter, and the resultant virus preparation is frozen until further processing into the vaccine.
• Roller bottles containing attached MRC-5 cells infected with varicella virus are rinsed, dispensed with 40 ml of either l .Ox or 1.5x PGSE stabilizer, and harvested from roller bottles by mechanically scraping the cell sheet from roller bottles using a robotic arm.
• the cell slurry is withdrawn from the roller bottle, collected in a vessel, and frozen to -60°C.
• an impinging jet apparatus Prior to processing, an impinging jet apparatus is calibrated to determine the pressure required to give the desired linear velocity, then sterilized.
• the vessels containing frozen harvested material are thawed, pooled, and placed in a pressure vessel connected to the impinging jet apparatus.
• the contents of the pressure vessel are pressurized to give a linear velocity through the jets of 22.5m/s ( 1 .Ox PGSE) or 25.0m/s ( 1.5x PGSE).
• the jetted material is transferred back in the pressure vessel for a second pass.
• two jets in series can be used.
• the disrupted cells are then clarified by filtration through polypropylene depth filters. Potency is assayed through a plaque assay. Particle size analysis using a Elzone particle analyzer is also completed.

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• 1996
  • 1996-10-01 TW TW085111939A patent/TW426733B/zh not_active IP Right Cessation
  • 1996-10-02 SK SK434-98A patent/SK43498A3/sk unknown
  • 1996-10-02 AR ARP960104582A patent/AR003776A1/es unknown
  • 1996-10-02 EP EP96933226A patent/EP0854913A1/en not_active Withdrawn
  • 1996-10-02 CZ CZ981041A patent/CZ104198A3/cs unknown
  • 1996-10-02 BR BR9610773A patent/BR9610773A/pt not_active Application Discontinuation
  • 1996-10-02 CN CN96198826A patent/CN1100868C/zh not_active Expired - Fee Related
  • 1996-10-02 EA EA199800266A patent/EA000703B1/ru not_active IP Right Cessation
  • 1996-10-02 WO PCT/US1996/015801 patent/WO1997012959A1/en not_active Application Discontinuation
  • 1996-10-02 AU AU72044/96A patent/AU7204496A/en not_active Abandoned

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