Patent Application
20170017891
The invention relates to a method of predicting fed batch production titer of a producer cell. In particular, the invention relates to a method of predicting relative fed batch recombinant protein titer of a panel of clonally derived producer cells.
Early prediction of the relative ability of CHO cell clonal isolates to serve as manufacturing cell lines is a key component of the cell line development process. Clones apparently productive in static culture can vary substantially in subsequent fed-batch culture performance, both with respect to cell growth and productivity. Current best practice is to measure clone performance early in cell line development using a small-scale, multi-parallel bioreactor system that enables comparison of 50-100 clonal isolates. However, this approach is still both costly and time-consuming.
It is an object of the invention to overcome at least one of the above-referenced problems.
The Applicant has discovered that the production titre of producer cell clones in fed batch culture can be accurately predicted by rapid profiling of clone-specific recombinant protein production response to one or more functionally diverse chemical stressors (for example inhibitors and toxins) in static microplate culture. To perform microplate profiling, a fixed number of clonally-derived cells were dispensed into 96-well microplates containing one or more chemical cell stressors separately loaded into discrete wells. Each chemical was soluble in cell growth medium and typically utilized at a concentration pre-determined to inhibit parental CHO cell proliferation in microplates within the LD30 to LD80 range. After static growth for a growth period (i.e. 3 days), supernatant MAb titer was measured by HPLC Protein A assay. Twelve CHO-S clones expressing an IgG1 MAb isolated by limiting dilution cloning were subjected to microplate profiling followed by assessment of optimized fed-batch culture performance (MAb titer [MAb], integral of viable cell concentration to 50% cell viability [IVCC50]) in Ehrlenmayer flasks. Using multi-linear modeling, it was demonstrated that clone performance in microplates could predict MAb titer (r2=0.84) using clone-specific MAb titer microplate profiles.
In a first aspect, the invention provides a computer-implemented method of predicting production titer of a query producer cell in fed batch culture, the method comprising the steps of:
In another aspect, the invention provides a computer-implemented method of predicting relative production titer of a panel of producer cells in fed batch culture, the method comprising the steps of:
When a batch of new clones are generated from a parental cell line, rather than all or a select few being subjected to expensive and time-consuming growth studies in bioreactors, the method of the invention allows a large number of clones to be rapidly assayed for, and ranked according to, predicted fed batch (recombinant protein) production titre in a rapid and high-throughput manner. This informs on which clones to take forward to scale-up/mini-bioreactor studies, for example only the good clones as illustrated in FIG. X below.
The methods of the invention employs data (production titer response profile) from a panel of pre-validated clones (clones of known production titer), and a computational model, ideally a multiple linear computational model, based on this data, to allow prediction of relative fed batch recombinant production titer of a new set of clones based on their rapidly obtained chemical fingerprints.
Preferably, the method comprises an additional step of ranking the clones according to predicted fed batch production titer.
The method is typically carried out using a microtitre plate, in which each assay is performed in a well of a microtitre plate. Suitably, the growth of each clonal cell is assayed in the well of a multiwell plate. When multiple chemical cell stressors are employed, each clone will be assayed in the presence of single chemical cell stressor.
Typically, the assay involves mixing a sample of a clone with a chemical cell stressor, and incubating the mixture from 1 to 4 days, typically 2 to 4 days, preferably 60-80 hours, and ideally about 3 days, and assaying the level of growth of the cells. Suitably, the clone sample is provided at a concentration of from 0.1 to 1.0×106 cells per ml of mixture. Typically, the bioreactor-relevant chemical cell stressor is provided at a concentration of 0.5 to 2×IC50, ideally about 1×IC50.
Suitably, the growth of each clone is assayed after a period of incubation of less than 5 days. Ideally, the growth of each clone is assayed after a period of incubation of between 1-4 days, and ideally after 2 and 3 days.
Preferably, the growth of each clone is assayed simultaneously. Typically, the incubation step is carried out in static microplate culture.
In a preferred embodiment, the invention provides a rapid, high-throughput, computer-implemented method of predicting relative production (i.e. monoclonal antibody) titer of a panel of clonal producer cells in fed batch culture, the method comprising the steps of:
The chemical cell stressors are chemicals that cause a reduction in cell growth via one or more of multiple cellular pathways. Suitably, the plurality of chemical cell stressors comprise at least 2, 3, 4, 5, or 6 functionally diverse chemical cell; stresspors, for example at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, or 25 different stressors. Typically, the plurality of chemical cell stressors are selected from the group consisting of amino acid transport inhibitors, cell cycle inhibitors, a source of osmotic stress, a source of oxidative stress, an inducer of apoptosis, metabolic effectors, a pH modifier, an inhibitor of glycolysis, and a toxin. These are examples of functionally diverse chemical cell stressors. Typically, the plurality of chemical cell stressors include stressors selected from at least 4, 5, 6 or 7 of the groups consisting of amino acid transport inhibitors, cell cycle inhibitors, a source of osmotic stress, a source of oxidative stress, an inducer of apoptosis, metabolic effectors, a pH modifier, an inhibitor of glycolysis, and a toxin.
In another aspect, the invention provides a microtitre plate comprising at least 24, 48 or 96 wells, and a plurality of chemical cell stressors disposed individually in at least some of the wells. Typically, the plate comprises at least 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, or 25 chemical cell stressors. Suitably, each chemical cell stressor is disposed in at least two or three wells of the plate (i.e. duplicate or triplicate).
Preferably, the plurality of chemical cell stressors are selected from the group of 2-aminobicyclo-(2,2,1)heptane-carboxylic acid (BCH), D-phenylalanine, α-(methylamino)isobutyric acid (MeAIB), Sodium Butyrate (NaBu), cycloheximide, ammonium chloride, Cadmium acetate dihydrate, Cobalt chloride (CoCl2), Sodium Chloride (NaCl), Sodium lactate (Na Lac), Aminotriazole (AMT), Menadione Sodium Bisulphite (MSB), Buthionine Sulfoximine (BSO), Mercaptosuccinic Acid (MS), 2,4,Dinitrophenol (24DNP), Sodium Oxamate, 2-deoxyglucose (2dg), 3-bromopyruvate (3-BrPA), Dichloroacetate (DCA), 6-diazo-5-oxo-1-norleucine (1-don), Valproic acid (Val), Sodium Orthovandate (NaV), citric acid, FK866, lactic acid.
In another aspect, the invention provides a microtitre plate comprising at least 24, 48 or 96 wells, and one or more, generally a plurality of, chemical cell stressors disposed individually in at least some of the wells, in which when a plurality of different chemical cell stressors is employed the chemical cell stressors comprise at least one chemical cell stressor selected from each of the groups consisting of amino acid transport inhibitors, cell cycle inhibitors, a source of osmotic stress, a source of oxidative stress, an inducer of apoptosis, metabolic effectors, a pH modifier, an inhibitor of glycolysis, and a toxin.
In one embodiment, the invention provides a microtitre plate comprising at least 96 wells, and at least 23 chemical cell stressors disposed individually in wells of the plate, in which the chemical cell stressors consist essentially of 2-aminobicyclo-(2,2,1)heptane-carboxylic acid (BCH), D-phenylalanine, α-(methylamino)isobutyric acid (MeAIB), Sodium Butyrate (NaBu), cycloheximide, ammonium chloride, Cadmium acetate dihydrate, Cobalt chloride (CoCl2), Sodium Chloride (NaCl), Sodium lactate (Na Lac), Aminotriazole (AMT), Menadione Sodium Bisulphite (MSB), Buthionine Sulfoximine (BSO), Mercaptosuccinic Acid (MS), 2,4,Dinitrophenol (24DNP), Sodium Oxamate, 2-deoxyglucose (2dg), 3-bromopyruvate (3-BrPA), Dichloroacetate (DCA), 6-diazo-5-oxo-1-norleucine (1-don), Valproic acid (Val), Sodium Orthovandate (NaV), citric acid, FK866, lactic acid.
In another aspect, the invention provides a kit suitable for performing a method of the invention and comprising (a) a microtitre plate of the invention (b) a microtitre plate reader, and (c) a computer program comprising program instructions for (i) receiving from a determination system (for example a HPLC) a production titre value of each clone in the presence and absence of the or each chemical cell stressor, (ii) calculating a normalised production titer value for each clone in the presence of the or each chemical cell stressor, (ii) inputting a clone-specific production titer response profile comprising the normalised production titer value for each clone in the presence of the or each chemical cell stressor into a computational model, in which the computational model is generated from production titer response profiles obtained from a calibration set of clones with known production titre values, wherein the computational model is configured to output the predicted fed batch production titre value for each clone (and optionally predict the relative fed batch production titer value of the panel of clonal cells), and (iii) outputting the predicted fed batch production titre value for each clone (and optionally predict the relative fed batch production titer value of the panel of clonal cells).
In another aspect, the invention provides a kit suitable for performing a method of predicting the relative fed batch production titre of a panel of clonal producer cells derived from a single parental host cell population and comprising (a) a microtitre plate of the invention (b) a microtitre plate reader, and (c) a computer program comprising program instructions for (i) receiving from a suitable machine a production titre value of each clone grown in the presence and absence of one or more chemical cell stressors, (ii) calculating a normalised production titer value for each clone in the presence of the or each chemical cell stressor, (ii) inputting a clone-specific production titer response profile comprising the normalised production titer value for each clone in the presence of the or each chemical cell stressor into a computational model, in which the computational model is generated from production titer response profiles obtained from a calibration set of clones with known production titre values, wherein the computational model is configured to output the predicted fed batch production titre value for each clone and predict the relative fed batch production titer value of the panel of clonal cells, and (iii) outputting the predicted relative fed batch production titer value of the panel of clonal cells.
The determination system employed to determine the production titer of a clone may be any suitable machine, for example a HPLC or mass spectrometer adapted to quantitatively assay the target recombinant protein.
The invention also provides a computer implemented system for performing a method of predicting fed batch production titer of a clonal producer cell derived from a single cell line, the system comprising:
The invention also provides a computer implemented system for performing a method of predicting relative fed batch production titer of a panel of clonal producer cells derived from a single cell line, the system comprising:
Typically, the determination system comprises a HPLC, although other systems adapted to quantitatively assay for a target protein, for example a quantitative ELISA, may be employed.
Suitably, the computational model is a multiple linear computational model. Ideally, the multiple linear computational model suitably employs a least squares solution to fit parameters (i.e. levels of cell specific production titer responses) to the observed fed batch production titer.
The invention also provides a computer program which when executed on a computer causes the computer to perform a method of predicting relative fed batch production titer of a panel of clonal cells derived from a single cell line according to the invention.
The invention also relates to a computer program recording medium storing a computer program according to the invention.
In one aspect, the invention relates to a method of screening a panel of producer cell clones derived from a single cell line to stratify the clones according to fed batch production titer. The method involves incubating a sample of each clone with and without one, and generally a plurality of, individual chemical cell stressors for a period of time, typically of at least 24 hours and up to 4 or 5 days, to obtain a plurality of normalised production titer response values for the clone. These production titer response value(s) provide a specific pattern of clone-specific production titer response that forms a clone-specific growth response profile. A multiple linear computational model is employed to correlate the production titer response profiles with a plurality of production titer response profiles from a calibration set of clones with know fed batch production titer, and predict a fed batch production titer for one or more of the panel of clones. The clones from the panel may then be ranked according to predicted fed batch production titer, which allows producer clones to be chosen for further development.
In this specification, the term “rapid, high-throughput” should be understood to mean that the method can be carried out in four days or less, and in which the incubation of cells during the incubation period may be carried out in the wells of a microtiter plate.
The term “production titer” or “production titer response”as applied to a specific producer cell clone refers to the amount of a specific protein, generally a recombinant protein, and ideally a recombinant monoclonal antibody, that the specific producer cell clone generates over a defined time period. The titer may be quantified in absolute or relative terms. Generally titre is referred to as weight of product per volume of culture—grams per litre (g/L) is a common metric (Max titer). Generally, the clones are incubated for a specific time period, for example 2-4 days, and following the incubation period a sample of the supernatant is typically taken and assayed for production titer. Various methods will be apparent to the person skilled in the art for measuring production titer, including HPLC and quantitative ELISA.
In many cases, the production titer employed in the methods of the invention will be normalised production titer response, meaning the difference between the production titer response for the cell when measured in the presence and absence of the chemical cell stressor.
The term “producer cell” refers to a cell that is employed to generate a specific desired protein. Generally, the cell is genetically modified to include one or multiple copies of a transgene encoding the desired protein which is generally under the control of a specific promoter. Thus, the specific protein is usually a recombinant protein. Producer cells are well known in the art, and include for example Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells. Ideally, the producer cell is a CHO cell. Typically, the producer cell is a monoclonal antibody producer cell.
The term “production titer response profile” refers to the production titer responses for a given clone to one or more chemical cell stressors. In one embodiment, the profile may include a single production titer response for a given clone (the production titer response for the cell in the presence and typically absence of one chemical cell stressor). In other embodiments, the production titer response profile comprises a plurality of production titer responses or a given clone (the production titer responses for the cell in the presence, and optionally absence) of a plurality of individual chemical cell stressor). Generally, the profile is generated using normalized production titer responses (production titer in absence of stressor minus production titer in presence of stressor), so that a normalized production titer response profile is obtained. However, in circumstances where the profile is generated using more than one stressor, it is possible to use profiles generated with production titres from stressed microenvironments only (i.e. without control production titres).
The method of the invention employs rapid profiling of producer cells to predict the production titer of the cells when in fed batch culture. In this specification, the term “fed batch” or “fed batch culture” should be understood to mean extended culture of cells where additional nutrients are added in bolus format at least once post seeding of the cells.
The term “calibration set of clones” refers to a plurality of clones, each having a production titer response fingerprint and a known fed batch performance. Typically, the calibration set of clones includes clones exhibiting a range of fed batch performance abilities from what the end user would consider ‘good’ performers to what the end user would consider ‘bad’ performers. Suitably, the calibration set of clones comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, or 26 clones
The term “stressed microenvironment” should be understood to mean an environment that includes a chemical cell stressor. Generally, this means that a clone is assayed for growth in a well of microtitre plate in the presence of the chemical cell stressor.
In this specification, the term “rapid” should be understood to mean a method of predicting fed batch performance that takes less than five days, and ideally less than four or three days.
In this specification, the term “high-throughput” should be understood to mean a method in which a large number of samples, for example 20-500, can be assayed simultaneously. In one embodiment, this involves assaying the growth of the clones in a multiwell plate, for example a 48, 96, or 192 well plate.
In this specification, the term “predicted fed batch production titer” as applied to a clonal producer cell should be understood to mean the predicted fed batch production titer of the clone in fed batch culture. The predicted production titer may be quantified in absolute terms, or may be quantified in relative terms, i.e. relative to a clone having known good or bad production titer or relative to a panel of clones.
In this specification, the term “predicting relative production titer” should be understood to mean predicting fed batch production titer of the clones in the panel relative to one another. Thus, in one embodiment, the clones are stratified according to their predicted fed batch production titer. Likewise, the term “predicted relative fed batch production titer of a panel of clonal cells” should be understood to mean the predicted fed batch production titer of the clones in the panel relative to one another. Thus, in one embodiment, the clones are stratified according to their predicted fed batch production titer. In another embodiment, the clones are stratified to pick one or more clones showing best predicted fed batch production titer clones, one or more clones showing worst predicted fed batch production titer clones, or both.
In this specification, the term “panel of clonal producer cells” should be understood to mean a panel of clonal producer cell populations derived from a single cell line, and comprising from 2 to 500 or more clonal producer cell populations. Methods for generating panels of clonal producer cells are well known to a person skilled in the art, and described in Production of recombinant protein therapeutics in cultivated mammalian cells (2004), Wurm, Florian M, New York, N.Y., Nature Biotechnology 22 (2004), S. 1393-1398. Typically, the panel of clonal producer cell populations include from 10-500, 20-500, 30-500, 40-500, 50-500, 60-500, 70-500, 80-500, 90-500 or 100-500 clonal cell populations. Typically, the panel of clonal producer cell populations include from 100-500, 100-400, 150-400, 150-350 clonal cell populations.
In this specification, the term “computational model” typically refers to a multiple linear computational model. Ideally, the multiple linear computational model employs a least squares solution to fit parameters (i.e. levels of cell specific production titer responses) to the observed fed batch production titer.
In this specification, the term “chemical cell stressor” should be understood to mean a chemical that can be incubated with a cell in a cell culture medium and is capable of causing a reduction in cell growth via one or more cellular pathways. Preferably, the chemical cell stressor is soluble in cell culture medium. Examples of chemical cell stressors (cell stressor types) include inhibitors of cellular pathways, cell toxins (chemicals that are toxic to cells, especially mammalian cells), metabolic effectors, and chemicals that stress the cells. Examples of inhibitors include amino acid transport inhibitors, cell cycle inhibitors, and glycolysis inhibitors. Examples of chemicals that stress cells are sources of osmotic or oxidative stress. Typically, the chemical cell stressor is utilised in an amount that inhibits cell proliferation within the LD30 to LD80 range. Suitably, the chemical cell stressor is utilised at a concentration of 0.5 to 2×IC50. Typically, a sample of each cell (i.e., a sample of each clone) is separately incubated with at least three different cell stressors. Preferably, a sample of each cell (i.e., a sample of each clone) is separately incubated with at least three different types of cell stressors (for example, an amino acid transport inhibitor, a glycolysis inhibitor, and a source of osmotic stress). Suitably, the plurality of chemical cell stressors comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, or 25 stressors. Typically, the plurality of chemical cell stressors are selected from the group consisting of amino acid transport inhibitors, cell cycle inhibitors, a source of osmotic stress, a source of oxidative stress, an inducer of apoptosis, metabolic effectors, a pH modifier, an inhibitor of glycolysis, and a toxin. Typically, the plurality of chemical cell stressors include stressors selected from at least 4, 5, 6 or 7 of the groups consisting of amino acid transport inhibitors, cell cycle inhibitors, a source of osmotic stress, a pH modifier, a source of oxidative stress, an inducer of apoptosis, metabolic effectors, an inhibitor of glycolysis, and a toxin. Examples of chemical cell stressors include:
BCH (AA transport Inhibitor)
D-phenylalanine (AA transport Inhibitor)
MeAIB (AA transport inhibitor)
Sodium Butyrate (Cell cycle inhibitor)
Cycloheximide (Toxin)
Ammonium chloride (Toxin)
Cobalt chloride (inducer of apoptosis)
NaCL (source of osmotic stress)
Sodium lactate (Metabolic effector)
Aminotraizole (source of oxidative stress)
Menadione sodium bisulphate (source of oxidative stress)
Mercaptosuccinic acid (source of oxidative stress)
2, 4 dinitrophenol (Metabolic effector)
Sodium oxamate (Glycolysis inhibitor)
2 deoxyglucose (Glycolysis inhibitor)
3 bromopyruvate (Glycolysis inhibitor)
Dichloroacetate (Glycolysis inhibitor)
L-don (AA synthesis inhibitor)
Valproic acid (cell cycle inhibitor)
sodium orthovandate (cell cycle inhibitor)
FK866 (apoptosis inducer)
citrate/lactate (pH modifier)
“IC50”: To establish inhibitory concentrations, “in house parental CHO” cells were incubated in 96 well plates in the presence of a range of concentrations of the chemical of interest for three days (72 hours). Growth was determined using a chemical viable cell number assay(“Presto Blue” from Life Technologies, UK). Growth in the presence of the chemical concentrations was compared to the control cell growth (i.e. in the presence of 0 mM inhibitory chemical) to produce a curve of chemical concentration vs percentage growth relative to the control. This curve was used to establish IC “X%” concentrations.
Exprimental
Cell Culture:
Clones used were CHO-S derived clones from a monoclonal antibody producing parental clone (clone 38) provided by Cobra biologics (Keele, UK). These are henceforth referred to as “PM-CHO”. PM-CHO Cells were cultured in CD-CHO media (Invitrogen, Paisley, UK), 8 mM L-Glutamine (Invitrogen, Paisley, UK), 1% HT supplement (Invitrogen, Paisley, UK) 12.5 μg/ml puromycin (Invitrogen, Paisley, UK). Cells were routinely sub-cultured on an alternating 3/4 day regime.
Fed Batch Studies:
Fed batch studies were performed in 60 ml volumes in shake flasks using commercially available feed and media. Feed was added in 6 ml boluses on days 4,6,8 and 10. Samples were taken daily and analysed for cell growth and monoclonal antibody titre.
Determination of Monoclonal Antibody Titre:
A HPLC method was employed using a “Biomonolith” protein A column (Aglient, Workingham, UK). Monoclonal antibody was quantified according to area under UV absorption peak in the eluent. This was demonstrated to be linearly proportional to antibody concentration in the sample.
Method of Performing Invention:
From a detailed literature survey, a variety of chemicals which were anticipated to be informative of fed batch performance were identified. These were:
Using an ‘in house’ parental cell line, inhibitory dose 50 (IC50) concentrations of the above chemicals were identified through growth studies. Here IC50 is defined as the concentration of the chemical, in question, which inhibits normal cell growth by 50% over a 3 day period. Once IC50 were established, 96 well plates were set up containing the above selection of chemicals at the IC50 concentration and including control wells which contained only cell growth media.
A panel of size 12 of monoclonal antibody producing clonal cell lines was generated in house by a limiting dilution cloning method using the PM parental cell line. These clones were subjected to fed batch studies to assess their fed batch product production ability. Here this is defined as the maximum volumetric product titre achieved during the fed batch run. The range of maximum titres achieved during fed batch by the clonal panel is illustrated in
Each clonal cell line was grown in a 96 well plate containing the above mentioned chemicals for 3 days. After this period the level of chemical specific product titre in the plates (i.e. MAb titre in the presence and absence of chemicals as mentioned above—normalized production titer) was measured using the “HPLC” assay as described above. This allowed a chemical specific product production fingerprint to be identified for each clonal cell line (referred to above as a clone specific production titer response profile). Here a product production fingerprint is defined as the plurality of product production levels in each chemical microenvironment.
The range of chemical specific productivities for the panel of clones is illustrated in
Using information from these chemical specific product production fingerprints, a multiple linear model was built using responses to individual chemicals as parameters.
A multiple linear model is defined as that satisfying the equation.
β=(XTX)−1XTy=(1/nΣxixiT)−1(1/nΣxiyi).
Where X represents the matrix containing appropriate data from the explanatory variables and Y is the vector of dependent (or response) variables. This is essentially finding a least squares solution to fit parameters (i.e. levels of chemical specific product production) to the observed fed batch performance (here defined as Max MAb titre).
An example of a multiple linear model built from the plurality of clone micro environments is
Max.MAb.Titre=37.78*(Control plate titre)−22.4150*(MeIAB plate titre)+0.4711
Where Max.Mab.Titre is the maximum monoclonal antibody titre achieved in fed batch culture. A graph illustrating the goodness of model fit using the above parameters is shown in
By using bootstrap cross validation techniques it can be shown that a model built in the above way can be predictive of future data, i.e. data not used in the model.
This model, build on a set of clones with known fed batch performance can use the ‘fingerprint’ of new clones and be able to predict their relative fed batch performance. Initially a model is generated using a panel of clones with known fed batch performance (in the above example 12 were used but there is no limitation to how many can be used—However the complexity of the model built is limited by the number of clones used). Subsequently when a large panel of clones is derived in the process of cell line development, these will be “screened” in the multiple microenvironment plates, and using the model generated from clones with known performance characteristics, it is possible to predict Max MAb titre, rank the clones or at least give information as to the likelihood of their future fed batch performance capabilities therefore aiding the process of selecting which clones are taken forward to the next stage of clones screening (i.e. small scale fed batch studies).
The embodiments in the invention described optionally comprise a computer apparatus and/or processes performed in a computer apparatus. However, the invention also extends to computer programs, particularly computer programs stored on or in a carrier adapted to bring the invention into practice. The program may be in the form of source code, object code, or a code intermediate source and object code, such as in partially compiled form or in any other form suitable for use in the implementation of the method according to the invention. The carrier may comprise a storage medium such as ROM, e.g. CD ROM, or magnetic recording medium, e.g. a memory stick or hard disk. The carrier may be an electrical or optical signal which may be transmitted via an electrical or an optical cable or by radio or other means.
In the specification the terms “comprise, comprises, comprised and comprising” or any variation thereof and the terms “include, includes, included and including” or any variation thereof are considered to be totally interchangeable and they should all be afforded the widest possible interpretation and vice versa.
The invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 14153333.1 | Jan 2014 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/078340 | 12/17/2012 | WO | 00 |
