WO2011047900A2

WO2011047900A2 - Novel process

Info

Publication number: WO2011047900A2
Application number: PCT/EP2010/062103
Authority: WO
Inventors: Jacques Dominique Marie Gerard; Isabelle Solange Lucie Knott
Original assignee: Glaxosmithkline Biologicals S.A.
Priority date: 2009-10-22
Filing date: 2010-08-19
Publication date: 2011-04-28
Also published as: WO2011047900A3

Abstract

The present invention relates to a process for detaching cells of an adherent cell type from particles of carrier which comprises the steps of: (a) providing in a vessel said cells adhered to particles of carrier in the presence of a growth medium, said vessel being characterised in that it comprises two internal zones, A and B, separated by a sieve, the sieve having an aperture size such that it is pervious to the cells if detached from the particles of carrier but substantially impervious to the particles of carrier, whereby the particles of carrier having cells adhered thereto are located within zone A but not zone B and zone B contains a drain opening under control of a valve adapted to permit vacation of liquid from zone A under an influence comprising gravity and/or pressure; (b) optionally washing the particles of carrier having cells adhered thereto by vacating liquid from the vessel via the drain opening in zone B under an influence comprising gravity and/or pressure and replenishing the vessel with another wash liquid and optionally repeating this step one or more times; (c) replacing the liquid in the vessel with a detachment buffer by vacating liquid from the vessel via the drain opening in zone B under an influence comprising gravity and/or pressure and replenishing the vessel with the detachment buffer; (c) detaching the cells from the particles of carrier by a process comprising an enzymatic detachment step performed in a detachment buffer.

Description

NOVEL PROCESS

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Aspects of this invention were made with United States government support pursuant to Contract # HHSO10020060001 1 C, from the Department of Health and Human Services; the United States government may have certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to a process for amplifying cells of an adherent cell type, in particular to a process involving a step of detaching adherent cells from a carrier, especially a particulate carrier. The process is useful in production of virus in such cells, especially influenza virus.

BACKGROUND OF THE INVENTION

Adherent cells are cultured in the production of recombinant proteins, recombinant viruses or naturally occurring viruses, so as to produce viral vaccines, for example. These cells must be grown on a surface, for example that of a particulate carrier such as Cytodex™ 1 , 2 and 3 (Pharmacia) microcarriers. Culturing adherent cells give rise to a number of well known difficulties. Firstly, the cell number is usually, restricted by available surface area, and the cell number is not normally expanded beyond that resulting from growth to confluence on the carrier. Although cells growing to confluence on a carrier will transfer to fresh cell-free carrier if this is added to the medium, this is an inefficient process. Consequently, for practical purposes, steps of detachment of cells from the carrier and reattachment to new carrier at lower density are required to achieve ongoing increase in cell density.

A common means of detaching cells from a carrier is by a process of trypsination involving treating cells with trypsin or a related proteolytic enzyme. In preparation for trypsination, steps of washing the cells and replacing the buffers are required. Following trypsination, the residual enzyme activity has to be lowered and this may be achieved by further steps of enzymatic inactivation by adding a trypsin inactivating agent, washing to eliminate the enzyme, or dilution with fresh media, so as to dilute the enzyme activity, so that the residual activity would be negligible. These steps may, optionally, be combined. Eventually a medium containing detached cells is produced which is suitable for seeding into a further culture vessel.

All the aforementioned washing and buffer replacement steps are time consuming which leads to the overall process being lengthy with consequent impact on production efficiency. Moreover the numerous manipulations can result in risk of contamination of the cell growth medium. There is a need to devise a process which is more efficient and ideally can simplify or accelerate the steps associated with cell detachment. This and other objects are addressed by the present invention.

Mundt (US5100799) describes a method for releasing cells from carrier particles comprising introducing a trypsin solution into a container containing cells immobilised on cell culture carrier particles in a continuous flow which is in a direction counter to gravity such that cells are released from the carrier particles and flow out of the top of the vessel with residual trypsin which is then inactivated. The container contains a permeable insert at a spaced location from its bottom whereby the carrier particles having cells immobilised thereto are located above the insert and a drain opening and the trypsin inlet are located below the insert.

Condon (US6783983) describes an apparatus for separating detached cells from carrier particles by use of a separation device in which the carrier particles are retained by a mesh having a mesh size which permits cells and aqueous solution to pass through.

Zhang (US2004/0058436) describes a cell detaching apparatus for detaching cells from carrier particles comprising a trypsinizing zone and a separating zone separated by a mesh, in which the separating zone is below the trypsinizing zone in the apparatus and in which the mesh size is between the diameters of the cells and the microcarriers.

None of the above apparatus or processes discloses an apparatus having a sieve with a surface orientated substantially vertically (rather than horizontally) or disclose an apparatus in which vacation of the reactant contents is facilitated by overpressure.

BRIEF SUMMARY OF THE INVENTION Thus, according to the invention, we provide a process for detaching cells of an adherent cell type from particles of carrier which comprises the steps of:

(a) providing in a vessel said cells adhered to particles of carrier in the presence of a growth medium, said vessel being characterised in that it comprises two internal zones, A and B, separated by a sieve, the sieve having an aperture size such that it is pervious to the cells if detached from the particles of carrier but substantially impervious to the particles of carrier, whereby the particles of carrier having cells adhered thereto are located within zone A but not zone B and zone B contains a drain opening under control of a valve adapted to permit vacation of liquid from zone A under an influence comprising gravity and/or pressure;

(b) optionally washing the particles of carrier having cells adhered thereto by vacating liquid from the vessel via the drain opening in zone B under an influence comprising gravity and/or pressure and replenishing the vessel with another wash liquid and optionally repeating this step one or more times; (c) replacing the liquid in the vessel with a detachment buffer by vacating liquid from the vessel via the drain opening in zone B under an influence comprising gravity and/or pressure and replenishing the vessel with the detachment buffer;

(d) detaching the cells from the particles of carrier by a process comprising an enzymatic detachment step performed in a detachment buffer.

The following steps may optionally thereafter be performed:

(e) treating the content of the vessel with an agent to inactivate the means of enzymatic detachment; and/or

(f) washing the detached cells and the particles of carrier by vacating liquid from the vessel via the drain opening in zone B under an influence comprising gravity and/or pressure and replenishing the vessel with another liquid free of any means for enzymatic detachment such that the detached cells are substantially retained within zone A and optionally repeating this step one or more times. BRIEF DESCRIPTION OF THE FIGURE

Figure 1 shows an example apparatus for use in the process according to the invention.

DETAILED DESCRIPTION

Description of the pressurisable cell detachment vessel

The vessel will typically comprise a portion with vertical walls (typically a cylinder) forming the sides, a lid and a base. The base portion may be substantially planar and positioned horizontally or may be conical, although suitably it is planar and horizontal. Typically the vessel will be constructed of robust materials, such as metals e.g. aluminium or stainless steel. The volume of the vessel may be adapted to the needs of the process to be employed, however commercial scale bioreactors are typically adapted to contain liquid volumes of 1 - 5000 litres, e.g. 10-100 litres such as 40-60 litres. The vessel comprises two zones, A and B, separated by a sieve. Suitably the sieve has a surface orientated substantially vertically. Most suitably the sieve is cylindrical and is positioned concentric with the central axis of the vessel whereby zone A is the zone formed within the interior of the sieve and zone B is the zone formed between the sieve and the side walls of the vessel. The sieve may be composed of metal, plastic or other suitable material for forming a classifying mesh or membrane. The aperture size will be such that it is pervious to the cells if detached from the particles of carrier but substantially impervious to the particles of carrier. "Substantially impervious" means that, typically, not more than 5% e.g. not more than 1 % e.g. not more than 0.1 % of particles of carrier (and, for example, no particles of carrier) are able to penetrate through the sieve. For example the aperture size of the sieve may be 50-150um e.g. 100um. The vessel is suitably pressurisable, typically it will be sealable, and will be adapted to tolerate pressure in use of up to, typically, 1 bar e.g. 0.1-1 bar such as 0.5 bar. Typically the vessel will be sterilizable. Application of pressure (e.g. overpressure to the headspace above liquid in the vessel) advantageously accelerates vacation of liquid from the vessel. Use of a sealed vessel and hence a closed system also avoids risk of contamination of the apparatus by airborne contaminants.

The vessel will desirably be provided with means for agitation of the content of zone A in the vessel. Suitably the means for agitation is one or more impellers which rotate about a shaft located along the central axis of the vessel.

The vessel will suitably be provided with a number of inlets and outlets as described in the foregoing. All inlets and outlets will suitably be provided with valves in order to permit the development of overpressure within the apparatus when needed, and to protect against contamination from external contaminants such as dust, pathogenic organisms and the like.

Liquid may be vacated from the vessel by means of a drain opening located within zone B. Suitably the line is located towards or at the base of the vessel.

The lid or upper portion of the vessel will suitably be provided with an inlet for air or other gas by which means to provide overpressure for the purpose of vacating the contents of the vessel and, if appropriate, to aerate the contents of the vessel (although aeration is not normally necessary during the trypsination step). The lid or upper portion of the vessel will suitably be provided with an escape valve for gas in case a threshold pressure is exceeded.

The vessel will suitably be provided with a temperature probe for the purpose of monitoring the temperature of the contents of the vessel. Suitably the temperature probe enters the vessel through the lid and along an axis parallel to the central axis within zone A.

The vessel will be suitably provided with a pH probe for the purpose of monitoring the pH of the content of the vessel. Suitably the temperature probe enters the vessel through the lid and along an axis parallel to the central axis within zone A.

The vessel will suitably be provided with one or more lines for the purpose of introducing liquid components into the vessel. For example one or more lines may be provided for the purpose of introducing buffers, growth media, acids, bases, enzymes (e.g. proteolytic enzymes) and the like into the vessel. Suitably these lines enter the vessel through the lid and along an axis parallel to the central axis within zone A.

The vessel will suitably be provided with one or more lines for the purpose of sampling the contents of the vessel. For example, a line may be provided for sampling liquid and/or cells and/or carriers from towards the bottom of the vessel or from the body of the vessel within zone A. A line may, in addition or instead, be provided for sampling liquid from zone B. The vessel will suitably be provided with one or more lines for the purpose of vacating the contents of zone A of the vessel including cells and carriers. Typically such a line would be provided towards the bottom of the vessel.

Carriers

Exemplary carriers on which cells are expected to grow are known in the art and preferably are adapted to the purpose of cell cultivation. The carrier is suitably a particulate carrier. Carriers may be made of any suitable material supporting cell growth, such as, but not limited to, dextran, plastic, gelatine, collagen or cellulose, glass or others as described in Butler (1988) Animal Cell Biotechnology, 3, 283-303, Spiers & Griffiths. They may be used uncoated or the surface of the carrier may be treated to modify cell adhesion, in particular to enhance cell adhesion yet permit proliferation and spreading, e.g. by coating such as with extracellular matrix proteins, such as collagen. Carriers may be of any shape and of any size. They are typically spherical, such as beads, or fibers made of synthetic or natural polymers or inorganic materials. They include microcarriers and macrocarriers. Microcarriers means that the carriers are microporous in the sense that cells cannot enter the carriers and only adhere to the surface of the carriers. Microcarriers are suitably spherical carriers, such as, for instance, the dextran Cytodex™ beads. Macrocarriers means that the carriers are macroporous in the sense that cells can enter the carriers. Such macrocarriers are suitably of a fibrous structure, such as BioNOCII™. They may also be of a spherical shape, such as the Cytopore™ beads, composed of cross-linked cotton cellulose. General reference is made to the book

"Microcarrier Cell Culture - Principles and Methods" published by Pharmacia. The minimum dimension of the carrier will typically be 100 μηη or more, say 130μ m or more. The maximum dimension of the carrier will typically be around 10 mm in the case of fibrous carriers.

Microcarrier beads of type Cytodex™ 1 , 2 or 3 are considered suitable. The particulate carrier typically has a particle size in the range of about 100-250 μηη, e.g. in the range 130-220 μηη and should be composed of a non-toxic material. The median of the sample size preferably falls within these ranges so that these size ranges represent at least the middle 90% of the carrier sample. Suitably the carrier consists of substantially spherical microbeads with a median particle size of about 150-200 μηη especially 170-180 μηη. Suitably the carrier particles are slightly denser than the culture medium such that they may be suspended by gentle agitation and such that they may be separated from the medium by sedimentation under gravity. A density of 1.03-1 .045 g/ml when the carrier particles are equilibrated with a standard solution such as 0.9% NaCI is considered suitable.

Suitably the carrier particle concentration for cell growth is between 1 and 20 g/L, e.g. between 5 and 15 g/L, e.g. around 9 g/L.

Cells The cells which are used in the method according to the invention can in principle be any desired anchorage-dependent (adherent) cell type which can be cultured in cell culture and, in particular, which can support virus replication. They can be either primary cells or continuous cell lines. Genetically stable cell lines are preferred. Mammalian cells are particularly suitable, for example, human, hamster, cattle, monkey or dog cells.

A number of mammalian cell lines are known in the art and include PER.C6, human embryonic kidney cells (293 cells), HeLa cells, CHO cells, Vero cells, MDCK cells and MRC5 cells. Suitable monkey cells are, for example, African green monkey cells, such as kidney cells as in Vero cell line. Suitable dog cells are, for example, kidney cells as in MDCK cell line. In case of growing influenza virus, particularly suitable mammalian cell lines include MDCK cells, Vero cells, or PER.C6 cells. These cell lines are all widely available, for instance, from the American Type Cell Culture (ATCC) collection. According to one embodiment, the method of the invention uses MDCK cells. The original MDCK cell line is available from the ATCC as CCL-34, but derivatives of this cell line may also be used. In a distinct embodiment, the method of the invention uses Vero cells.

Alternatively, adherent cell lines for use in the invention may be derived from avian sources, such as chicken, duck, goose, quail or pheasant. Avian cell lines may be derived from a variety of developmental stages including embryonic, chick and adult. In particular, cell lines may be derived from the embryonic cells, such as embryonic fibroblasts, germ cells, or individual organs, including neuronal, brain, retina, kidney, liver, heart, muscle, or

extraembryonic tissues and membranes protecting the embryo. Chicken embryo fibroblasts (CEF) may be used.

Culture of cells

The cells that are provided in step (a) are adhered to particles of carrier and have been cultured in a growth medium, generally in a separate bioreactor. When the cells are cultured in a separate bioreactor, they are aseptically transferred to the detachment vessel according to the present invention immediately prior to detachment.

Cells are suitably seeded into the bioreactor at a density of around 0.1-1 x 10⁶ cells/ml e.g. around 0.375 x 10⁶ cells/ml and are cultured, typically, until they are substantially confluent. By "confluent" is meant that the cells have formed a coherent monocellular layer on the surface of the carrier, in particular, a particulate carrier, so that virtually all available surface is used. By "substantially confluent" is meant that, typically, at least 70% e.g. at least 80% or 90% or 95% of the available surface of the carrier is used. Here, available surface means sufficient surface area to accommodate a cell. Therefore small interstices between adjacent cells that cannot accommodate an additional cell do not constitute "available surface". Whether or not the cells have reached confluence may be determined by sampling. A small sample of carrier particles may be sampled via a sampling line and viewed under a microscope.

Cells may be grown in a variety of cell culture media which may be serum free - such as Iscove's medium, ultra CHO medium (Lonza), EX-CELL (SAFC Biosciences), Ultra MDCK (Lonza), OPTIPRO (Invitrogen) or a conventional serum containing medium such as MEM or DMEM with a proportion (e.g. 0.5-10%) of fetal calf serum. Serum free media are preferred.

Cells may be grown in the bioreactor in batch mode, fed-batch or perfusion mode. Detachment (trypsination) is normally conducted when the cells are substantially confluent and maximum cell density is reached. Cell growth rate may vary according to the cell type which is cultured. Therefore, the time needed for a cell culture to reach confluency, i.e. the culture time which will elapse before proceeding to trypsination, may vary according to each cell type. As a non-limiting example may be cited the MDCK cells for which typically a cell density of at least 5 x 10⁶ cells/ml e.g. around 5 - 6 x 10⁶ cells/ml is targeted to be reached after around 5-6 days of cell culture e.g. after 5 days of cell culture, if 0.375 cells/ml were initially seeded. According to one embodiment, trypsination according to the method of the invention is performed once cell density reached 5 x 10⁶ cells/ml. However, the method of the invention is suitable for trypsinizing any cell culture at any desired cell density and does not require that the cell density be maximal before trypsination. For instance, trypsination may be performed before cells reached confluence, in particular, trypsination may take place while cells occupy less than 90%, e.g. less than 80 or less than 70% of the available surface.

Washing (step (b))

The contents of the vessel are vacated by releasing the drain opening in the vessel and allowing the liquid contents of the vessel to empty under an influence comprising gravity and/or pressure. Typically the vessel empties under the influence of gravity with assistance through application of overpressure to the headspace above the liquid in the vessel. The reduction of volume leads to concentration of the carrier particles. However due to the arrangement of the sieve, having a vertically orientated surface, concentration of the carrier particles does not lead to blockage of the sieve apertures which is advantageous, especially when pressure is applied. In order to avoid drying of the cells, emptying of the contents is stopped when a substantial proportion of the liquid contents (e.g. 75% or more e.g. 80% or 90% or more) has been removed. The drain opening may be closed, and replacement medium (such as a wash liquid such as PBS optionally together with components such as glucose and/or EDTA) may be added. The process may be repeated any number of times, depending on the extent of washing desired. Typically two or three washing steps are performed.

Although it is preferred to perform the wash step in batch mode, as just described, in principle it could be performed in continuous mode (this would be less efficient, however). In a continuous mode, the replacement liquid is introduced into the vessel at the same time as the original liquid is vacated from the vessel. The same comment applies to any of the wash or liquid/buffer replacement steps described below.

Replacement of liquid with detachment buffer (step (c))

The liquid in the vessel may be replaced with a buffer appropriate for the detachment step (d) by following a step essentially similar to the step described above as step (b). Thus a substantial proportion of the liquid contents of the vessel is vacated via the drain opening (e.g. 75% or more e.g. 80% or 90% or more) and is replaced by detachment buffer. To enhance the efficiency of buffer replacement, step (c) may be performed one or more times, or step (b) may be performed in which the wash buffer is the detachment buffer.

Adhesion of cells to the carrier is promoted by alkaline earth metal salts such as calcium and magnesium salts. Therefore, the detachment buffer will suitably not contain any components which promote cell adhesion and, for example, alkaline earth metal salts such as calcium and magnesium salts will suitably be avoided. A chelating agent such as EDTA or citrate may suitably be included in the detachment buffer. A suitable buffer is PBS containing glucose (1 g/L) and EDTA (0.1-0.2 g/L) (the glucose component is optional).

Detachment (step (d))

In principle cells may be detached from a carrier to which they are adhered by a number of well known enzymatic means. The most common means of detachment is using proteolytic degradation, most typically employing a cysteine or serine endopeptidase. In one embodiment the enzyme is derived from a plant, such as a cysteine endopeptidase e.g.

papain, actinidin, bromelain or ficin. In another embodiment the enzyme is a protease derived from a bacterium or fungus. In another embodiment the enzyme is a recombinant enzyme e.g. a mammalian protein recombinantly expressed in a bacterium or fungus. Use of a plant, bacterium or fungus derived enzyme or a recombinant enzyme, for example a mammalian enzyme or a derivative thereof expressed in a bacterium or fungus is preferred in order to avoid use of products of animal origin. Suitably the enzyme is trypsin or a trypsin-like proteolytic enzyme. A suitable enzyme is TrypLE (Invitrogen) which is a trypsin like enzyme based on a mammalian trypsin together with a signal sequence from the fungus Fusarium oxysporum expressedin the fungus Fusarium venenatum. See WO2004/020612 for further details. Another example is Trypzean, a recombinant trypsin from Sigma. The concentration to be used is in accordance with manufacturer's directions. The activity of enzymes, in particular tryspin enzymes are typically expressed in USP/ml (Unit Specific Protease/ml). However, the activity of some of them, such as TrypLE, may also be expressed as rpu/ml (Recombinant Protein Unit/ml). Both unit types can be easily converted one to the other according to the following formula: 1 rpu = 293 USP. Typically the enzyme will be employed at a concentration of around 30-100 USP/ml. The progress of the detachment step can be monitored by sampling carriers from the vessel and assessing under the microscope whether cells remain adhered to the carriers or not. The step is considered complete when carriers in the sample as viewed under the microscope are substantially free of adhered cells, for example after around 10-60 minutes. The duration of the detachment step may be dependent on different factors, such as the cell type, as some cells may be more easily detached than others, and the enzyme concentration.

Inactivation (step (e))

In order to prevent unwanted reaction of the reagents used in the detachment step, optionally an inactivating agent can be added to neutralise the detachment reagents. Thus when the detachment agent is trypsin or a similar proteolytic enzyme, the inactivating agent could be a chemical or proteinaceous enzyme inhibitor (such as an irreversible active site directed chemical inhibitor). More suitably the inactivating agent is a proteinaceous trypsin inhibitor such as soybean trypsin inhibitor (STI).

Washing (step (f))

This optional step is conducted in substantially the same way as step (b). The washing step allows the reagents associated with detachment to be removed from the medium in which the detached cells are suspended. The replacement liquid may be a wash liquid (such as PBS) or a cell growth medium. The advantage of the apparatus of the invention in this step is further apparent because since the sieve is vertically and not horizontally orientated, the apertures do not become blocked by the detached cells.

Optional further steps

In order to achieve further passages of cell growth, typically cells (optionally together with any carrier particles with which they are associated) will be sampled from the vessel at the end of steps (d), (e) or (f) (these cells may be considered as the primary culture) and seeded into a further vessel at lower density in the presence of further carrier particles where they may again be cultured to increase cell number (this culture may, then, be considered as the secondary culture). A non-limiting typical seeding size is 0.1-1 x 10⁶ cells/ml e.g. 0.375 x 10⁶ cells/ml. The seeding size may vary from one cell type to another. The passage from a primary culture to a secondary culture is characterized by a split ratio which represents the proportion of the primary culture in the form of detached cells which is required for seeding the further vessel at a given cell density and providing thus the secondary culture. The desired split ratio when passaging cells is usually within the range of 1/4 to 1/15.

If neither of optional process steps (e) and (f) is performed, it will be necessary to lower the proteolytic enzyme activity by dilution with the addition of fresh culture medium to the trypsinized cells used for seeding the further culture vessel. The volume of fresh medium may vary, in particular, according to the concentration of the trypsin which has been used for trypsination. Typically, the volume of fresh medium which is added ranges from 20% to 80%, e.g. 30% to 60%, around 40% of the seeding volume.

Growth in secondary culture suitably results in a cell density value, x days post- trypsination, which is similar to the cell density value reached by the primary cell culture x days post-seeding. Indeed, in these conditions similar cell density values for the primary and the secondary cultures indicate that the secondary culture, i.e the culture after trypsination, did not suffer any harm from the trypsination and presents similar growth characteristics, such as cell viability and ability to proliferate. For example, trypsinizing a primary cell culture which reached a cell density of around 5 x 10⁶ cells 5 days after seeding according to the method of the invention provides a secondary cell culture reaching a cell density of at least 5 x10⁶ cells e.g. around 5 - 6 x 10⁶ cells/ml 5-6 days after trypsination.

General process parameters

Preferably the process, and all reagents, buffers and other media used in it, is entirely free of products of animal origin.

Steps of the process will typically be conducted under appropriate temperature control. A temperature probe may be provided in the device for this purpose. Typically cell culture steps are conducted at or around 37 °C. Typically cell detachment by proteolytic enzymes is conducted at or around 37 °C.

Steps of the process will typically be conducted under appropriate pH control. A pH probe may be provided in the device for this purpose. Typically cell culture and washing steps are conducted at pH 7.0-7.8 e.g. at or around pH 7.3. Typically cell detachment by proteolytic enzymes is conducted at or around pH 7.6. Since the trypsination step is normally undertaken at a slightly higher pH than the washing steps, it will usually be necessary to add base to the medium at or around the time of adding the trypsin or trypsin like enzyme.

Process steps will generally take place with agitation, typically at 60-1 Orpm.

Utility of the invention

Adherent cells may be cultured for the purpose of producing recombinant proteins. Mammalian cell types are especially preferred for this purpose. Adherent cells may also be cultured for the purpose of producing live virus for use in production of vaccines. Live virus may be used as such or, more commonly, processed in certain ways to reduce or eliminate pathogenicity e.g. by heat, cold or radiation treatment, partial purification, virion splitting, or sub-unit isolation. A number of viruses may be produced in this way, including

Orthomyxoviridae, such as influenza virus, Paramyxoviridae, including RSV, mumps and measles, Picornaviridae, such as polio, and HAV, Reoviridae, such as rotavirus, Flaviviridae, such as dengue, Herpesviridae, such as varicella, Rhabdoviridae, such as rabies, Togaviridae, such as Rubella, Coronaviridae, such as SARS, or Caliciviridae, such as Norwalk. In particular, the method of the present invention is suitable for producing influenza virus. A method of growing influenza virus in adherent cells is described in Brown

(US4500513), the contents of which are herein incorporated in their entirety. In summary, cells are grown to confluence on carriers, the growth medium is removed, and replaced with a new medium containing live virus working seed. The infected cells are incubated, typically at 32- 37°C for 1-72 hours. Efficiency of influenza virus production is enhanced by addition of a proteolytic enzyme, such as trypsin, after cells were infected. The infected cells are further incubated, typically at 32-37°C, until maximum viral yield is achieved, typically after 3 to 6 days. Further aspects of the invention include:

A process for culturing cells of an adherent cell type which comprises the steps of:

(i) providing in a vessel said cells adhered to particles of carrier in the presence of a growth medium, said vessel being characterised in that it comprises two internal zones, A and B, separated by a sieve, the sieve having an aperture size such that it is pervious to the cells if detached from the particles of carrier but substantially impervious to the particles of carrier, whereby the particles of carrier having cells adhered thereto are located within zone A but not zone B and zone B contains a drain opening under control of a valve adapted to permit vacation of liquid from zone A under an influence comprising gravity and/or pressure, and whereby the cells are grown such that they are substantially confluent on the particles of carrier;

(ii) optionally washing the particles of carrier having cells adhered thereto by withdrawing liquid from the vessel via the drain opening in zone B under an influence comprising gravity and/or pressure and replenishing the vessel with another wash liquid and optionally repeating this step one or more times;

(iii) replacing the liquid in the vessel with a detachment buffer by withdrawing liquid from the vessel via the drain opening in zone B under an influence comprising gravity and/or pressure and replenishing the vessel with the detachment buffer;

(iv) detaching the cells from the particles of carrier by a process comprising an enzymatic detachment step performed in a detachment buffer;

(v) optionally treating the contents of the vessel with an agent to inactivate the means of enzymatic detachment;

(vi) optionally washing the detached cells and the particles of carrier by withdrawing liquid from the vessel via the drain opening in zone B under an influence comprising gravity and/or pressure and replenishing the vessel with another liquid free of any means for enzymatic detachment such that the detached cells are substantially retained within zone A and optionally repeating this step one or more times; and (vii) transferring a sample of detached cells and, optionally, any associated carriers to another vessel for further cell growth.

A process for virus production which comprises (a) culturing cells according to the aforementioned process and allowing the cells to grow to confluence on the particles of carrier, (b) infecting said cells with virus; and (c) harvesting the virus;

A process for virus production according to the aforementioned process wherein the efficiency of virus production is increased by treating the cells infected with cells with a proteolytic enzyme prior to harvesting;

A process for vaccine production which comprises producing a virus according to the aforementioned process and preparing a vaccine from said virus;

Such a process for vaccine production wherein the virus is processed to reduce or eliminate its pathogenicity prior to vaccine production;

Such a process wherein the virus is influenza virus; and

A vaccine producable by the aforementioned processes.

There is also provided a cell detachment vessel (for example a pressurisable vessel) comprising two internal zones, A and B, separated by a sieve, the sieve having an aperture size such that it is pervious to the cells if detached from the particles of carrier but substantially impervious to the particles of carrier, whereby the particles of carrier having cells adhered thereto are located within zone A but not zone B and zone B contains a drain opening under control of a valve adapted to permit vacation of liquid from zone A under an influence comprising gravity or pressure, further characterised in that the sieve has a surface orientated substantially vertically. There is also provided such a vessel containing adherent cells and particles of carrier having cells adhered thereto. There is also provided such a vessel containing a liquid cell growth medium. There is also provided such a vessel containing a detachment buffer. There is also provided such a vessel containing a detachment buffer and trypsin or a trypsin like proteolytic enzyme.

A further aspect of the invention is the use of such a vessel in a process for the detachment of adherent cells from a carrier, such as from a particulate of carrier. A further aspect of the invention is the use of such a vessel in a process for culturing cells e.g. for culturing cells infected with virus such as influenza virus.

EXAMPLES

The invention will now be illustrated by reference to Figure 1 in which a vessel is provided (shown in cross section from the side) having cylindrical walls, a base and a lid. Inside the vessel a cylindrical sieve (1 ) is positioned concentric with the central axis. The sieve defines two zones - a zone A forming the interior of the vessel and zone B between the sieve and the walls of the vessel. The sieve may have, for example, 100 μηη apertures. Agitation is provided by impellers driven by a rotor positioned along the central axis. Various inlets are provided including buffer/medium inlet (3), air inlet (4), detachment reagent inlet (5), acid/base addition inlet (8) and various outlets are provided including air outlet (pressurisation valve) (6), transfer outlet line (7), sampling line (9), bottom sampling line (1 1 ) and drain opening (12). Various probes are provided including a pH probe (2) and a temperature probe (10).

Example 1 : Trypsination method using a trvpsinizer device

Overview:

Trypsination was performed in a stainless steel vessel as illustrated in Figure 1 , 50L size with ca. 40L working volume, with an internal 100 μηη sieve. MDCK cell culture grown on microcarrier beads (Cytodex 1 , Pharmacia, 9 g/L) and considered as the primary culture were transferred from a bioreactor to the trypsination vessel under aseptic conditions.

Media and buffers were removed during two washing steps comprising filtering the contents of the vessel through the sieve under overpressure of 0.5 bar.

Trypsin like enzyme (TrypLE, Invitrogen) was added.

Growth medium was added to dilute the enzyme activity, so that the residual activity, if any, in the final volume is negligible, in the sense that it will not prevent subsequent cell re-attachment of detached cells to new microcarriers during subsequent cell growth.

An inoculum of detached cells from the trypsination vessel was transferred to a secondary bioreactor, providing thus the secondary culture.

The following criteria were measured to assess the efficiency of the technology:

-The total cell recovery which represents the percentage of cells detached from the

microcarrier beads, as compared with the number of cells present on the microcarriers before starting washing the cells, reflecting thus, in particular, the detachment efficiency.

-The cell viability which represents the percentage of cells that are alive after trypsination.

-The cell density reached by the secondary culture a few days after tryspination.

-The split ratio representing the proportion of detached cells from the primary culture which is required for seeding the next bioreactor with 0.375 x 10⁶ cells/ml.

-Process duration

Methods

1. The protocol evaluated with the trypsinizer device:

Table 1 : Trypsination protocol

2. Cell culture parameters in further bioreactor to assess the impact of trypsination conditions on subsequent cell growth. Table 2: Subsequent cell growth parameters

Secondary culture in

bioreactor

Bioreactor B-Braun Working volume 5L

Seeding size 0.375 10⁶ cells/ml

Seeding mode Dilution

Cytodex 1 concentration 9 g/L

Culture medium In house cell culture medium

Agitation 60 rpm

3. Results and conclusions

A primary culture of MDCK cells grown on microcarriers Cytodex 1 beads (9 g/L) was performed at 80L scale until the desired cell density was reached. Half of the culture volume (40L) was transferred to the trypsination device when the cell density was 5 x 10⁶ cells/ml (Test 1 of Table 3 and Table 4), while the other half was left grown until the cell density reached 8.5 x 10⁶ cells/ml (Test 2 of Table 3 and Table 4) before being transferred to the trypsination device. Tryspination was performed according to the general protocol as described in Table 1 and according to the specific conditions as detailed in Table 3.

Table 3: Bead-to-bead transfer process according to trypsination protocol tested with trypsinizer device.

The efficiency of trypsinizer device is measured in terms of process duration, cell recovery and ability of the secondary culture to grow. The ability of the secondary culture to grow is determined by measuring the cell density (cells/ml) at day 5 or 6 post-trypsination and comparing it to the cell density of the primary culture measured at day 5 or 6 post-seeding. Having similar numbers means that trypsination did not impact the ability of the cells originating from the primary culture to grow, after trypsination, as the secondary culture. A decrease in that number, i.e. having a lower cell density after trypsination, for the secondary culture, reflects a negative impact of trypsination on the cells originating from the primary culture impaired in their ability to grow as the secondary culture. The results are summarized in Table 4.

Table 4: Process results obtained according to process tested on trypsinizer device.

These results show that:

· The trypsination process via the device according to the invention can be performed in about 2 hours.

• Growth of cells in secondary culture is good (cell density of more than 5 x 10⁶ cells/ml after 5 days with a cell viability between 79 and 87 %). The split ratio (>1/5) and total cells recovery (25 to 41 %) are acceptable. Example 2: Comparative Example - Trypsination method by settling

A trypsination method was performed without using a trypsinizer device. Trypsination was performed directly in a 40 L bioreactor comprising the MDCK culture grown on microcarrier Cytodex™ 1 (9 g/L) beads for 5 or 6 days, according to the following conditions:

- The microcarrier beads were settled (settling took between 10 to 15 minutes).

- Culture medium was removed.

- Washing buffer comprising PBS/EDTA 180 mOsm/l was added to the beads for 20 minutes (pH 7.3, 37°C and 50 rpm stirring).

- The microcarrier beads were settled (settling took between 10 to 15 minutes).

- Washing buffer was removed.

- Washing buffer comprising PBS/EDTA 220 mOsm/l was added to the beads for 45 to 80 minutes (pH 7.3, 37°C and 50 rpm stirring).

- The microcarrier beads were settled (settling takes between 10 to 15 minutes).

- Washing buffer was removed.

- TrypLE enzyme-containing buffer (100 USP/ml) was added to the beads for 30 to 45 minutes (pH 7.6, 37°C and 50 rpm stirring).

- Fresh culture medium was added to the trypsinized beads to inactivate the trypsin by dilution (around 40% of fresh medium, as compared to the trypsination volume).

Two different cultures in two different bioreactors were trypsinized according to the above scheme (Test 1 and test 2 of Table 5). Duration of the steps and of the full process, as well as cell recovery, and ability of the secondary culture to grow were measured. The results are summarized in Table 5.

Table 5: Results obtained according to the trypsination method by settling

Results - Comparison of Table 4 and Table 5 - The two processes involving use of a trypsinizer device (Table 4) or trypsinizing the cells directly in the culture bioreactor (Table 5) give similar results for (i) the total cell recovery, i.e. 41 and 25% (Table 4) versus 44% and 33% (Table 5), (ii) the cell viability, i.e. 87% and 79% (Table 4) versus 95% and 85% (Table 5), and (iii) the ability of the secondary culture to grow (see last rows of Tables 4 and 5, where cells density values are indicated). The trypsination impact on the cell density value of the secondary culture measured 5 or 6 days after trypsination (to be compared to the cell density values obtained for the primary culture) is similar whether the trypsinizer device is used (Table 4) or not (Table 5).

- However, the total duration of the process is significantly different in the two processes. If trypsinizing a similar volume (40 L) of culture, the process using the trypsinizer device according to the invention is approximately half the duration of the process performed without using the device (settling method), i.e. 130 min and 105 min (Table 4) versus 246 and 247 min (Table 5). In particular, when using the trypsinizer device, the trypsination step can be shortened up to 4.5 times (see Table 4: 10 min versus Table 5: 45 min. The shortest possible trypsination time is desirable to minimise the contact time between the trypsin and the cells in order to avoid undesired proteolytic activity which may impact cell viability and subsequent cell growth. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

All references referred to in this application, including patents and patent applications, are incorporated herein by reference to the fullest extent possible as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

Claims

WHAT IS CLAIMED IS:

1. A process for detaching cells of an adherent cell type from particles of carrier which comprises the steps of:

(b) optionally washing the particles of carrier having cells adhered thereto by withdrawing liquid from the vessel via the drain opening in zone B under an influence comprising gravity and/or pressure and replenishing the vessel with another wash liquid and optionally repeating this step one or more times;

(c) replacing the liquid in the vessel with a detachment buffer by withdrawing liquid from the vessel via the drain opening in zone B under an influence comprising gravity and/or pressure and replenishing the vessel with the detachment buffer;

2. A process according to claim 1 which comprises one or more of the following steps performed after step (d):

(e) treating the contents of the vessel with an agent to inactivate the means of enzymatic detachment; and/or

(f) washing the detached cells and the particles of carrier by withdrawing liquid from the vessel via the drain opening in zone B under an influence comprising gravity and/or pressure and replenishing the vessel with another liquid free of any means for enzymatic detachment such that the detached cells are substantially retained within zone A and optionally repeating this step one or more times.

3. A process according to claim 1 or claim 2 wherein the vessel is a pressurisable vessel and wherein in one or more (and preferably all) of steps (b), (c) and (f), liquid is vacated from the vessel through application of overpressure to the headspace above the liquid in the vessel.

4. A process according to any one of claims 1 to 3, wherein the cells are MDCK cells.

5. A process according to any one of claims 1 to 3, wherein the cells are Vero cells.

6. A process according to any one of claims 1 to 5, wherein the cells of step (a) are substantially confluent on the particles of carrier.

7. A process according to any one of claims 1 to 6, wherein sieve has apertures of size 50-150 m.

8. A process according to any one of claims 1 to 7, wherein the overpressure applied in optional step (b) and in steps (c) and (f) is between 0.1 and 1.0 bar.

9. A process according to any one of claims 1 to 8, wherein the process for detachment of step (d) comprises use of trypsin or a trypsin like enzyme.

10. A process according to any one of claims 1 to 9, wherein the detachment buffer of step (c) is free of alkaline earth metal salts.

1 1. A process for culturing cells of an adherent cell type which comprises the steps of:

(i) providing in a vessel said cells adhered to particles of carrier in the presence of a growth medium, said vessel being characterised in that it comprises two internal zones, A and B, separated by a sieve, the sieve having an aperture size such that it is perviousto the cells if detached from the particles of carrier but substantially impervious to the particles of carrier, whereby the particles of carrier having cells adhered thereto are located within zone A but not zone B and zone B contains a drain opening under control of a valve adapted to permit vacation of liquid from zone A under an influence comprising gravity and/or pressure, and whereby the cells are grown such that they are substantially confluent on the particles of carrier;

(iii) replacing the liquid in the vessel with a detachment buffer by withdrawing liquid from the vessel via the drain opening in zone B under an influence comprising gravity and/or pressure and replenishing the vessel with detachment buffer;

(vi) optionally washing the detached cells and the particles of carrier by withdrawing liquid from the vessel via the drain opening in zone B under an influence comprising gravity and/or pressure and replenishing the vessel with another liquid free of any means for enzymatic detachment such that the detached cells are substantially retained within zone A and optionally repeating this step one or more times; and

(vii) transferring a sample of cells and any associated carriers to another vessel for further cell growth.

12. A process for virus production which comprises (a) culturing cells according to claim 1 1 and allowing the cells to grow to confluence on the particles of microcarrier, (b) infecting said cells with virus; and (c) harvesting the virus.

13. A process for virus production according to claim 12, wherein the efficiency of virus production is increased by treating the cells infected with cells with a proteolytic enzyme prior to harvesting.

14. A process for vaccine production which comprises producing a virus according to claim 12 or claim 13 and preparing a vaccine from said virus.

15. A process according to claim 14, wherein the virus is processed to reduce or eliminate its pathogenicity prior to vaccine production.

16. A process according to any of claims 12 to 15, wherein the virus is influenza virus.

17. A process according to any one of claims 1 to 16, wherein the process, and all reagents, buffers and other media used in it, is entirely free of products of animal origin

18. A vaccine producable by a process according to any of claims 14 to 17.

19. A cell detachment vessel comprising two internal zones, A and B, separated by a sieve, the sieve having an aperture size such that it is pervious to the cells if detached from the particles of carrier but substantially impervious to the particles of microcarrier, whereby the particles of carrier having cells adhered thereto are located within zone A but not zone B and zone B contains a line under control of a valve adapted to permit withdrawal of liquid from zone A when overpressure is applied to the contents of the vessel, further characterised in that the sieve has a surface orientated substantially vertically.

20. A vessel according to claim 19 which contains (i) adherent cells and particles of carriers having cells adhered thereto in a liquid cell growth medium; or (ii) adherent cells and particles of carriers in a detachment buffer optionally further containing trypsin or a trypsin like proteolytic enzyme.

21. Use of a vessel according to claim 19 or 20 in a process for detachment of adherent cells from a carrier, such as from particles of microcarrier.

22. Use of a vessel according to claim 19 or 20 in a process for culturing cells e.g. cells infected with virus such as influenza virus.