HK1157387B - Use of hsa-producing cells - Google Patents

Description

Use of HSA-producing cells

Technical Field

The present invention relates to the field of cell culture technology and to methods for replicating/cloning cells, preferably cell lines, which are important for the production of biopharmaceuticals. The invention also relates to methods of preparing proteins using the obtained and replicated cells, and to media compositions in which individual cells may be replicated.

Background

The market for biopharmaceuticals for the treatment of humans is rapidly growing around the world. More than 900 biopharmaceuticals are currently being tested in clinical trials, and it is estimated that in 2010 there may be a renewal to 500 billion (Datamonitor 2007).

Currently, more and more biopharmaceuticals are produced in mammalian cells because of their ability to properly process and engineer human proteins. Therefore, the successful production of high yields of biopharmaceuticals in mammalian cells is of critical importance and depends on the characteristics of the recombinant monoclonal producer cell line used in the manufacturing process. In addition, the time course of cell line development is a critical time factor, which is a concern about how long a biological agent can enter clinical trials. In view of these aspects, there is an urgent need to accelerate and make more efficient the cell line opening process.

For biotechnological production of biologically active or therapeutic proteins (so-called biopharmaceuticals) in mammalian cells, the corresponding mammalian cell line is stably transfected with DNA encoding the biologically active protein (or subunit thereof). After this transfection process, a population (millions) of differently transfected cells is typically obtained. The key step in the preparation of efficient production cell lines is therefore the selection and replication of cell clones which, on the one hand, grow very stably and, on the other hand, exhibit a high specific productivity of the therapeutic protein (product formed, etc.). Since there are millions of different product expressing cells, it is critical to be able to analyze a plurality of high throughput cells individually and use automation to be able to sort out suitable candidates (single cell clones) that grow very vigorously and also produce high product titers. This single cell isolation and transfer process is called cloning or recloning.

Animal cell cultures are used to produce cell cultures for which genotypic and phenotypic homology (i.e., monoclonals) is desired for the biopharmaceutical. This can be achieved by re-cloning techniques such as "limiting dilution" or by automated storage of individual cells by Fluorescence Activated Cell Sorting (FACS).

However, there are problems in efficiently replicating typical recombinant production cells such as mouse myeloma cells (NSO), hamster ovary Cells (CHO), or hamster kidney cells (BHK), particularly when the cells are adapted to be grown in serum-free suspension cultures (i.e., under modern production-related cell culture conditions), whereby the cells are separately deposited in microtiter plates under serum-free culture conditions after recloning.

This result is due to the fact that in vivo cells are embedded in the tissue matrix and supply autocrine and paracrine growth factors by neighboring cells. Thus, if the cells do not gradually adapt to the new conditions, the cells do not adapt to the split growth and die without growth factor stimulation.

In particular, the use of serum-free or chemically defined media in the recloning step results in limited recloning efficiency, i.e., only a small fraction of the deposited cells survive and grow into a monoclonal cell line.

The "limiting dilution" and FACS re-cloning techniques currently used are well known in the art.

In "limiting dilution", the cell suspension is serially diluted and subsequently placed into a microtiter plate at different cell numbers per well. In each well containing a large number of cells, many or all of the cells survive by fully secreting autocrine growth factors. The fewer cells seeded per well, the fewer surviving cells, so the dilution can be adjusted in such a way that statistically only one cell per well survives and grows into a monoclonal cell line. These individual cell clones are detected by visual and/or imaging techniques and grown in larger culture vessels.

In FACS techniques, a flow cytometer is used to generate single cell clones. To this end, the cells are placed in a laminar flow and individually into each well of a microtiter plate. This ensures that the surviving colonies are in fact independent clones. Therefore, FACS techniques are preferred over limiting dilution.

The use of serum-free or chemically defined media in the recloning step results in limited recloning efficiency, i.e. only a small percentage of the individually placed (einzeln abegelten) cells grow into a monoclonal cell line.

Thus, at low recloning efficiencies, many microtiter plates must be packed into single cells to obtain the desired number of independent clones, which is time consuming and expensive (e.g., in terms of media, petri dishes, etc.).

Low recloning efficiency is particularly disadvantageous if subsequent analysis of the single cell clones is to be performed using an automated system. An analytical robot cannot normally discriminate between wells containing live or dead cells, but automatically measures all wells of the microtiter plate. When the recloning efficiency is only 10%, this means that in 90% of the cases the robot analyzes the empty wells and the living cell clones are analyzed using the same amount of reagents. Thus, in this example, 90% of the time and 90% of the material cost are wasted without obtaining any data.

To solve this problem, serum (e.g., fetal calf serum, FCS) has often been added to the culture medium in the past. Serum contains an undefined mixture of different soluble proteins and growth factors that maintain cell survival and proliferation. However, for regulatory reasons, the use of non-defined additives such as serum is increasingly poorly tolerated, due in part to the risk of infection with bovine virus. Thus, in general, serum-free production of cell lines is state of the art from a regulatory point of view.

Another possible solution is to perform a "limiting dilution" on the re-cloning. Since this method only statistically produces single cell clones but many clones can also grow in one well, this process must be repeated several times (typically 2-3 times) to ensure that the obtained cell line does in fact originate from only a single clone. These repeated cycles involve high labor and time costs, which have a negative impact on the required costs and time lines.

In the other method, "Feeder" cells (Feeder Zellen) are used. The name comes from the english word "feed", meaning that it is usually co-cultured with non-dividing cells that are required to be fed in culture with nutrients and secretory growth factors. The efficiency of recloning may be significantly improved by the feeder cells.

In another approach, recombinant proteins are added to the recloning medium to promote survival and growth of the individual deposited cells. Examples of recombinant proteins used are insulin, insulin-like growth factor (IGF), Epidermal Growth Factor (EGF) or Human Serum Albumin (HSA). These additives are obtained as purified proteins or protein solutions and added to the culture medium. Disadvantages of this process are the high cost, the dependence on the availability of recombinant proteins and their instability. Such drawbacks include, among others:

the need to store the recombinant proteins at-70 ℃,

freeze/thaw cycles inactivate the recombinant proteins and must therefore be avoided,

rapid loss of activity in solution under incubation conditions.

The object of the present invention is to increase the recloning efficiency of serum-free FACS-based producer cell clones.

Summary of The Invention

A solution to this technical problem of low recloning efficiency for serum-free media, preferably chemically defined media and/or insulin-free media, is obtained by using cells producing transgenic albumin or HSA or by using media conditioned by such HSA-producing cells. Thus, HSA is not added to the medium from the outside as a purified protein but is released directly from the living cells into the recloning medium.

The following examples are possible:

1. HSA-producing cells were used to prepare conditioned Medium (konditinierten Medium) for the periodic storage of single cells.

2. HSA-producing cells are used as feeder cells. Compared to the use of recombinant HSA added at once to the culture medium, the feeder cells produce HSA continuously in multi-well plates after storage of single cells and thus contribute to an increased growth time of the stored cells (compared to a single addition of recombinant protein).

3. HSA-producing cells are used as host cells for the production of biopharmaceuticals. Thus, the cells are able to promote their growth by autologous secreted HSA even in the absence of feeder cells and recombinant additives.

The highest recloning efficiency can preferably be achieved by a combination of these methods, i.e.by using HSA-producing host cells and HSA-conditioned medium. Depending on the circumstances and on the characteristics of the subsequent analysis methods, feeder cells may additionally be used.

By using HSA-producing cells as host cells or as feeder cells or as medium regulators, the recloning efficiency and the number of clones thus obtained can be significantly increased. This effect was observed even at concentrations of the cloning medium below 200mg/L HSA, even <100 mg/L. The activity/efficacy of secreted HSA on the recloning efficiency is thus significantly improved compared to recombinant HSA which is typically added at concentrations of 400mg/L to >2 g/L.

A preferred embodiment uses HSA-producing cells in a medium that does not contain insulin.

The positive effect of HSA-producing cells and/or their culture medium on the efficiency of recloning was evident in different media both in the presence and in the absence of feeder cells.

This is also evident in different cell lines/cell classes such as, for example, hamster and mouse cells, as well as in humans and various other cell systems.

HSA-producing cells used can be naturally HSA-producing cells (e.g., human liver cells) as well as genetically engineered cells of other species.

A preferred embodiment consists of a transgenic HSA-producing production cell line, such as CHO, BHK, NS0, Sp2/0 or Per.C 6 cells. A particularly preferred embodiment comprises HSA-producing CHO cells.

The methods described herein for increasing the efficiency of recloning in serum free media by using HSA-producing cells or media conditioned thereby can achieve more efficient clone screening and thereby more efficient cell line development procedures.

In biopharmaceutical development, the main goal of recloning is to recognize high-yielding cell clones. Higher efficiency of re-cloning (achieved by the large number of clones obtained and the normal distribution of the clones in terms of productivity) increases the likelihood of obtaining highly productive cell clones.

Significant time and labor and, therefore, cost savings are also possible because fewer cells must be deposited in a plate at higher re-cloning efficiencies to obtain the same number of viable cell clones. The process steps are therefore overall more economical.

Furthermore, the improvement of the recloning efficiency is based on the use of an efficient automated clone screening method (screening automaton). In such systems, individual wells are typically not selected in a microtiter plate, but all wells are measured equally, regardless of whether the wells contain a population of living cells. Since all wells of the well plate were analyzed, the number of clones examined increased in proportion to the number of clones exhibited. A recloning efficiency of only 10% means that 90% of the wells were empty and therefore unable to provide any usable data but still analyzed. In terms of economy, this means that 90% of the cases spend time and money (in reagent form) without obtaining any data. Thus, the efficiency of a high throughput automated system for early clonal selection is increased in proportion to the number of clones obtained after single cell storage.

Another advantage of the method is that the addition of HSA-producing cells as host cells and/or as medium regulators can increase the recloning efficiency to such an extent that feeder cells are no longer needed.

On the one hand, this reduces the manpower involved in the production and pre-incubation of feeder cells and at the same time increases the reproducibility of single cell clones. In addition, this makes it possible to use techniques for visualizing the number of viable cells as a tool for clonal analysis. Since the amount of product in the culture medium is salty-lined with the number of producing cells, the technical development sought how to detect both the amount of product and the number of cells in order to determine from these the highest producing cell clones (i.e.those with the highest specific productivity).

Since it is difficult to visually distinguish between feeder cells and emerging producer cells, these feeder cells are also included in the visual cell number, leading to inaccurate calculation of the total cell number and thus inaccurate determination of specific productivity-critical parameters. Thus, a method that does not involve feeding cells is advantageous and represents a preferred embodiment.

Possible applications of the method are found in particular in the production of biopharmaceuticals.

Increasing the efficiency of recloning to produce producer cells for the production of biopharmaceuticals increases the likelihood of producing highly productive clones and thus improves or accelerates product development.

The method also improves the economics of this step and makes it possible to use high throughput automated clonal analysis techniques.

The addition of recombinant proteins to insulin-containing media for the purpose of increasing the efficiency of recloning has indeed been described in the prior art (WO 2006047380). In contrast, however, the use of secreted albumin or HSA, or albumin-producing cells or HSA-producing cells in culture medium is explicitly described in the present patent application as an alternative and good possibility. HSA is not exogenously added to the medium as a recombinant purified protein but is secreted directly into the re-cloning medium by cells transgenic for albumin.

HSA-producing CHO cells are indeed described in the literature but have no description of the use of HSA-producing cells as host cells for the production of proteins other than HSA or for their use as feeder cells or as medium regulators for increasing the efficiency of recloning.

Drawings

FIG. 1: possible applications of the invention

Schematic representation of possible embodiments of HSA producing cells:

(1) for preparing conditioned Medium

(2) As feeder cells

(3) HSA-producing cells are used as host cells for biopharmaceutical production, i.e.cells producing the protein of interest additionally secrete HSA and thereby promote their own growth during recloning.

These possible applications can be combined together in order to increase the reclosing efficiency even further.

FIG. 2: conditioned medium for preparing HSA-producing cells to increase re-cloning efficiency

(A) This figure shows the relative recloning efficiency when using fresh recloning medium without any additions (negative control, white), and the increase in relative recloning efficiency when adding 500mg/L recombinant HSA (positive control, grey), or the increase in relative recloning efficiency of 50% conditioned medium from a culture of HSA-producing cells and removed from the culture and added after the indicated days (black band).

(B) The summary of the HSA concentrations measured in the above-mentioned medium is tabulated.

Maximum recloning efficiency was obtained when using medium conditioned for 2-5 days. It is significantly higher than the recloning efficiency when recombinant HSA was added to the medium, but the HSA concentration was quantitatively determined to be reduced by 5-50 fold in the conditioned medium. Any longer term regulation, although increasing the amount of HSA in the medium, also leads to a decrease in recloning efficiency.

FIG. 3: re-cloning efficiency is increased by using HSA-conditioned medium

(A) Effect on HSA-conditioned medium in the presence of feeder cells. This figure shows the relative recloning efficiency of cells deposited individually on 10,000 growth arrested feeder cells. The negative control showed the recloning efficiency (set here as 1) of the Bellingger's own medium without any additives, while 500mg/L of recombinant HSA was added to the medium in the positive control. Since a conditioned medium containing HSA-producing cells cultured for 2 days was used, the recloning efficiency could be further improved. The HSA concentration of the medium was measured to be higher than that of the higher bands.

(B) Influence of addition of HSA-conditioned medium to a commercial medium (HyQ SFM 4CHO medium). The graph shows the recloning efficiency of a medium different from the medium in (A) when no feeder cells were used. In this medium, no individual deposited cells grow into a viable culture in the medium without additives. A few cells survived due to the addition of recombinant HSA (500mg/L, dark grey band). On the other hand, higher recloning efficiency (light grey band) was achieved by using conditioned medium of HSA producing cells. The bands are evaluated for the average of 3-5 times for each condition of the 96-well plate; error bars show standard deviation.

FIG. 4: increased efficiency of recloning by HSA-producing feeder cells

Single cells from a cell population are deposited by either autologous CH-DG44 feeder cells or transgenic HSA-producing feeder cells. The recloning efficiency using DG 44-cells was set to 1 (black band). The grey band gives a relative increase in recloning efficiency when using HSA-producing feeder cells. The bands indicate the average of 3 evaluations of the 96-well plate for each condition.

FIG. 5: combination of HSA-producing feeder cells and host cells

This figure shows the efficiency of recloning in batches containing untransfected DG44 cells deposited on autologous feeder cells (1 placement, black) when the HSA-producing cells were recloned in the presence of DG 44-feeder cells (grey). This combination (white) of HSA-producing cells deposited on HSA-transgenic feeder cells further significantly improved the efficiency of re-cloning. The graph shows the mean of 3 assessments on a 96-well plate, with error bars showing standard deviation.

FIG. 6: concentration-dependent increase in recloning efficiency due to addition of recombinant HSA

This figure shows the recloning efficiency when recombinant HSA was added at the indicated concentration. In this experiment the maximum efficiency was achieved when 1000mg/L recombinant HSA was added to the re-cloning medium; this value is set to 100% and the measured efficiencies of other batches are recorded relative to the set value.

Detailed description of the preferred embodiments

Definition of

Before the present invention is described in greater detail by way of the following non-limiting illustrative examples, it is noted that the use of indefinite articles such as "a" or "an" and definite articles such as "the" include both the singular and the plural of such words unless specifically excluded in either of the singular and the plural forms and unless a specific form (singular or plural) is mentioned. Thus, the term "cell" automatically also includes "cells" unless it is explicitly stated that only a single cell is intended. It will be explicitly pointed out that in the singular, for example, the references to "a" or "an" are supplemented by (1).

The term "insulin" indicates a growth factor secreted by pancreatic beta cells. Insulin is known to those skilled in the art. Human insulin zinc or recombinant insulin are often used in cell culture. The insulin concentration of the medium can be measured using routine experiments such as a commercially available insulin-specific ELISA.

The term "insulin-free" means that the medium does not contain any insulin, in particular, does not contain any recombinant insulin and also does not have insulin added thereto.

Albumin is the most common protein in plasma. It is produced in the liver and contributes to the maintenance of osmotic pressure in the blood. Albumin binds to nutrients and metabolites and thus facilitates their transport.

The term "albumin" in the present invention indicates a polypeptide component having the biological activity of albumin. Albumin is generally an animal albumin, specifically, a mammalian albumin such as, for example, human albumin, bovine albumin, equine albumin, murine albumin, rat albumin, and porcine albumin, and an avian albumin such as, in particular, chicken albumin. Preferably, the albumin is Human Serum Albumin (HSA). As used herein, the terms "HSA" and "albumin" are synonymous, i.e., "HSA" refers to human albumin and albumin from other animal species. In the present invention, it should be clearly distinguished between recombinant HSA added to the medium from the outside and "secreted HSA" released directly (i.e., without purification or isolation) from HSA-producing living cells into the re-cloning medium. When albumin or HSA secreted directly into the cell culture medium by live cells of the trans-albumin gene is mentioned, this means that albumin or HSA is not added from the outside as a purified protein.

The production of recombinant HSA is well known in the art and can be performed using, for example, genetically engineered yeast (U.S. Pat. No. 5,612,197). Recombinant HSA is commercially available from various suppliers, for example, Sigma-Aldrich (recombinant HSA, catalog number A-7223). This is a purified protein that can be added to the incubation medium.

The data of the present invention show that "secreted HSA" has significantly higher activity/efficacy compared to HSA, since it can significantly improve re-cloning efficiency at significantly reduced concentrations.

According to the invention, the term "significantly reduced concentration" means:

-the concentration is reduced at least two-fold compared to recombinant or purified albumin,

-a concentration reduction of at least five times compared to recombinant or purified albumin,

-the albumin concentration is reduced at least 2-fold compared to a comparable culture mixture containing recombinant or purified albumin,

-the albumin concentration is reduced at least 5-fold compared to a comparable culture mixture containing recombinant or purified albumin,

-the albumin concentration is reduced at least 2 to 100 fold, 2 to 5 fold, 5 to 100 fold, 20 to 100 fold, 5 to 10 fold compared to a comparable culture mixture containing recombinant or purified albumin,

an albumin concentration of less than 200mg/L, preferably less than 100mg/L, particularly preferably less than 60 mg/L.

In contrast to recombinant HSA, secreted HSA is released directly from HSA-producing cells in live culture into re-cloning medium. It can be derived from 3 sources: (a) HSA-producing cells from the primary culture used to prepare the conditioned medium, (b) HSA-producing feeder cells, or (3) HSA-secreting host cells (when the individual deposited producer cells are genetically engineered to secrete HSA themselves).

The concentration of HSA can be determined by conventional methods such as commercially available ELISA (e.g., "human albumin ELISA quantitation kit", Bethy Laboratories, Montgomery, TX).

The terms "albumin or HSA transgenic cell", "albumin or HSA producing cell" or "albumin or HSA secreting cell" indicate any cell that releases albumin into the culture medium. This may be a cell that naturally secretes albumin (e.g., a liver cell) or a cell that is genetically engineered, for example, by introducing a construct that expresses albumin or HSA such that it expresses albumin or HSA and releases it into the culture medium. A preferred embodiment of the invention consists of albumin-or HSA-producing mammalian cells, most preferably albumin-or HSA-producing rodent cells, especially albumin-or HSA-producing CHO-or NSO-cells. Designated terms such as "albumin-or HSA transgenic cells", "albumin-or HSA producing cells" and "albumin-or HSA secreting cells" are used interchangeably in this application.

The "specific productivity" of a cell indicates the amount of a particular protein produced per unit time (i.e., released into the culture medium) by a cell in the case of secreted proteins. Specific productivity is calculated from the concentration of the product in the medium (= titer, measured by e.g. ELISA) and the number of producer cells present over the time span under consideration (also referred to as "IVC", integral of the number of live cells over time). Specific productivity is usually given in "pcd" (= pg/cell day = picograms of protein secreted per cell per day).

The terms "cloning/re-cloning", "cloning/re-cloning" in relation to cell culture mean a technique by which a cell population of homogeneous cells can be obtained from the original cells. Thus, the term "cell clone" or "single cell clone" means a process of: wherein single cells can be identified and isolated from a cell population of cells having different genotypes and subsequently replicated to form a cell population consisting of a plurality of genetically identical cells. If the cells are stored individually, i.e., only one (1) cell per culture vessel and subsequently expanded to form a cell population of homogeneous cells, the process is a "direct single cell clone". If a plurality of cells are simultaneously stored in one culture vessel, expanded to form a cell population, and this can be divided into cell populations of the same kind of cells by repeated dilution (= limiting dilution), this is described as an "indirect cloning" method.

"Single clone" or "single cell clone" or simply "clone" is derived from a genetically identical cell of (1) single cells. Thus, a cell population consisting of cells of the same origin and species is hereinafter referred to as a "monoclonal cell population". For the purposes of the present invention, each individual cell of this cell population is still considered to be a homogeneous cell and the culture is considered to be a monoclonal cell population, provided that there are several spontaneous changes (e.g., mutations and/or translocations) in the genome during the cultivation of the cells of the same origin. In contrast, a population of stably transfected cells (transfectants) is not a clone of cells of a different lineage, i.e., it is not a monoclonal cell population, even though starting cells of the same gene are transfected with the same nucleic acid.

The term "subclone/subculture" refers to different generations of cells produced from a primary cell or primary culture by single or multiple passage of dividing cells. For example, the word "subclone/subculture" is used when a homogeneous cell or cell culture is grown and replicated for many generations.

The term "cloning efficiency" or "recloning efficiency" is defined as the percentage of cells that can survive, divide and form an extremely important cell population after storage. For example, if 100 cells are distributed in 100 culture vessels in a cell sorting operation and if 25 of the 100 individually deposited cells grow to form a culture, the cloning efficiency is 25%.

By "effective or efficient recloning" is meant a cloning efficiency of at least 10%, preferably at least 20%, more preferably at least 30% and even more preferably at least 40%. According to a particularly preferred embodiment of the present invention, the term "efficient recloning" means a cloning efficiency of at least 50%, preferably at least 60%, optimally at least 70% and even more preferably at least 80%.

For the purposes of the present invention, the term "capable of dividing/expanding" describes the potential possibility of an endless but at least 2, preferably 4, passage division of a cell/cell population. This potential can be reduced or completely destroyed by, for example, irradiation with [137] Cs or treatment with mitomycin C.

The term "derivative/progeny" refers to cells that can be traced back on a gene to a particular starting cell and formed, for example, by transfection (with or without selective pressure) and/or produced by gene manipulation. The re-isolation of cells of the same cell type is also encompassed by the term "derivative/progeny". Thus, for example, all CHO cell lines are derivatives/progeny of hamster ovary cells isolated from the white hamster (Cricetulus griseus) (Puck et al, 1958), regardless of whether they are obtained by transfection, re-isolation or genetic manipulation.

The term "feeder cells" from the english word "feed" refers to cells that are typically co-cultured with desired non-dividing cells for feeding in a culture with nutrients and secretory growth factors. For preparation, living cells may develop growth arrest by irradiation with UV or gamma rays or treatment with mitomycin C. The resulting feeder cells live and produce and secrete growth factors but are unable to divide further.

The term "autologous feeder cells" means that both feeder cells and cells that are cultured in the presence of the feeder cells are taxonomically derived from the same source. For example, in case the cell line to be cultured is a hamster cell (hamster subfamily (cricetneae)) and preferably a cell of the genus cricke (Cricetulus) or crickes (Mesocricetus), e.g. a CHO or BHK cell, each feeder cell derived from this subfamily is an autologous feeder cell for such hamster cells belonging to the hamster subfamily.

According to a preferred embodiment, the term "autologous feeder cells" means feeder cells and cells to be grown that are taxonomically derived from the same species or originally isolated from the same species (cells from the hamster or whole hamster). For example, in case the cell to be cultivated is a hamster cell of the hamster or of the whole hamster, preferably a CHO or BHK cell, each feeder cell is initially isolated from said species as an autologous feeder cell within the meaning of the invention.

According to another preferred embodiment, autologous feeder cells are present if the feeder cells and the cells to be cultured are from the same species (e.g., grey-binned or whole-binned mice). According to a particularly preferred embodiment, autologous feeder cells (e.g., ovarian cells-CHO cells from grey stores) are present, provided that the feeder cells and the cells to be cultured are from the same species and have the same tissue tropism.

According to a particularly preferred embodiment, the feeder cells are autologous feeder cells, provided that the feeder cells and the cells to be cultivated originate from the same basic cell, e.g.provided that both cells ultimately are CHO-DG-44 cells or progeny of these cells. According to another preferred embodiment, the feeder cells and the cells to be cultivated are equally resistant, e.g. to antibiotics. This is particularly advantageous when cell storage is performed in the presence of a selection agent (e.g., an antibiotic).

In one aspect, the invention features the use of HSA-producing feeder cells for increasing the efficiency of recloning. In a preferred embodiment, HSA-producing autologous feeder cells are used.

The term "limiting dilution" indicates an alternative method of recloning. The cell suspension was serially diluted and the cells were then stored in microtiter plates at different cell numbers per well. In wells with high cell numbers, many or all cells survive due to sufficient secretion of autocrine growth factors. The fewer cells seeded per well, the fewer surviving cells, which means that dilution can be adjusted accordingly so that statistically only one single cell per well survives and grows into a monoclonal line. Since this method only statistically forms single cell clones, but there may be several clones growing in one well, this method must be repeated several times (typically 2 to 3 times) in order to ensure that the obtained cell line is actually from a single clone.

The term "conditioned medium" means a medium derived from a culture of living cells. The action of the conditioned medium is based on its content of growth factors which are secreted into the medium by the cells of the preliminary culture and thus have the effect of "regulating" the medium.

Thus, the terms "Medium-binding element (Zellen)" or "Medium-binding element (Zellen)" refer to cells used to prepare conditioned Medium.

In one aspect, the invention describes the use of HSA-producing cells as medium regulators so that the conditioned medium thus obtained contains, inter alia, HSA secreted by the cells.

The term "serum" refers to the cell-free component of blood. Serum contains a defined mixture of different soluble proteins and growth factors that contribute to cell survival and proliferation. For cell culture, Fetal Calf Serum (FCS) or bovine serum (FBS) is mainly used. When added to the culture medium, the concentration range is usually 10-20% FCS or FBS.

The term "serum-free" means a medium and incubation conditions characterized by the growth of cells in the absence of animal and/or human serum, preferably in the absence of any protein isolated from serum, preferably in the absence of non-recombinantly produced protein. Thus, the term "serum-free conditioned cells" means cells that replicate in the absence of serum or serum proteins in an animal or human.

The term "protein-free" means that the culture medium does not contain any animal proteins; proteins isolated from bacteria, yeast or fungi are not considered animal proteins.

The term "chemically defined" describes a cell culture medium which is serum-free, preferably also protein-free, and which is composed of chemically defined substances. The chemically defined medium thus consists of a mixture of predominantly pure individual substances. An example of a chemically defined medium is CD-CHO medium produced by MessrsInvitrogen (Carlsbad, CA, US).

"cells that can be cultured in suspension" refers to cells that are adapted to grow in liquid culture ("suspension culture") and have limited or lost their ability to adhere to the surface of a container (e.g., a cell culture dish or flask). Cells adapted to grow in the absence of serum and adapted to grow in suspension are referred to as "non-adherent cells adapted to serum-free medium". If feeder cells are prepared from these cultures, these cells are defined as "non-adherent feeder cells adapted to serum-free medium".

The term "protein/product of interest" refers to a protein/polypeptide having significant biological activity, including, for example, antibodies, enzymes, cytokines, lymphokines, adhesion molecules, receptors, and derivatives or fragments thereof. However, the target protein/product is not limited to these examples. In general, all polypeptides that are useful as agonists or antagonists and/or have therapeutic or diagnostic utility are significant or related. Other proteins of interest are, for example, proteins/polypeptides which can be used to alter various properties of the host cell within the scope of so-called "cell engineering", such as, but not limited to, anti-apoptotic proteins, chaperones, metabolic enzymes, glycosylases, and derivatives or fragments thereof.

The term "polypeptide" is used with respect to an amino acid sequence or protein and refers to a complex of amino acids of any length. The term also includes proteins that have been engineered post-translationally by reactions such as glycosylation, phosphorylation, acetylation, or protein processing. The structure of a polypeptide can be engineered, for example, by substitution, deletion, or insertion of amino acids and fusion with other proteins while retaining their biological activity. In addition, the polypeptides may be multimerized and combined to form homo-and heteromers.

By recombinant protein is meant a protein that can be produced by recombinant expression in a host cell. The recombinant protein lines are produced under the most stringent purity conditions in order to minimize the risk of contamination. Recombinant proteins are typically produced in suitable host cells such as, for example, yeast cells, animal cells, or prokaryotic cells (e.g., E.coli or other strains) using expression vectors such as, for example, plasmids, phages, naked DNA, or viruses to introduce the recombinant protein into the host cell. Recombinant proteins are generally available in purified form as concentrated protein solutions or in powder form. Recombinant HSA is available from various commercial suppliers such as Sigma Aldrich, for example.

Examples of therapeutic proteins are insulin, insulin-like growth factors, human growth hormone (hGH) and other growth factors, receptors, tissue plasminogen activator (tPA), Erythropoietin (EPO), cytokines, e.g., Interleukins (IL) (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18), Interferons (IFN) - α, - β, - γ, - ω, or- τ, Tumor Necrosis Factors (TNF) (e.g., TNF- α, - β, or- γ), TRAIL, G-CSF, TNF-gamma, GM-CSF, M-CSF, MCP-1, and VEGF. Other examples are monoclonal, polyclonal, multispecific, and single chain antibodies and fragments thereof (e.g., Fab ', F (ab ')2, Fc, and Fc ' fragments), light (L) and heavy (H) chain immunoglobulins and constant, variable, or hypervariable regions thereof, as well as Fv and Fd fragments (Chamov et al, 1999). The antibodies may be of human or non-human origin. Humanized and chimeric antibodies are also possible.

Fab fragments (antigen binding fragment = Fab) are composed of the variable regions of two chains held together by adjacent constant regions. It can be produced, for example, by treatment of conventional antibodies with proteases such as papain or by DNA cloning. Other antibody fragments are F (ab')2 fragments, which can be produced by proteolytic digestion with pepsin.

Short antibody fragments consisting only of the variable regions of the heavy (VH) and light (VL) chains can also be prepared by gene cloning. These are called Fv fragments (variable fragment = fragment of the variable part). Since covalent bonding by cysteine groups of the invariant chain is not possible in these Fv fragments, the fragments are usually stabilized by some other method. For this purpose, the variable regions of the heavy and light chains are typically joined together by a short peptide fragment of about 10 to 30 amino acids, preferably 15 amino acids. This results in a single polypeptide chain with VH and VL linked together by a peptide linker. Such antibody fragments are also known as single chain Fv fragments (scFv), examples of ScFv antibodies are known and described, see, e.g., Huston et al, 1988.

Over the past few years, various strategies have been developed for producing numerous scFv derivatives. The intention is to produce recombinant antibodies with improved pharmacokinetic properties and enhanced binding affinity. To achieve diversity of scFv fragments, the scFv fragments are produced as fusion proteins with multiple fusion domains. The consensus domains may be, for example, the CH3 region of IgG, or a helical structure ("coiled coil structure"), such as a leucine Zipper (Leucin-Zipper) domain. In other strategies, multimerization is performed using interactions between the VH and VL regions of scFv fragments (e.g., bi-, tri-, and penta-specific antibodies).

The term bispecific antibody (diabody) is used in the art to refer to bivalent homodimeric scFv derivatives. shortening of the peptide linker group in the scFv molecule to 5 to 10 amino acids results in VH/VL chain overlap to form homo-isomers. The bispecific antibodies may additionally be stabilized by inserted disulfite bridges. Examples of bispecific antibodies can be found, for example, in Perisic et al 1994.

The term minibody (minibody) is used in the art to indicate a bivalent homo-dimeric scFv derivative. It is composed of a fusion protein containing the CH3 domain of an immunoglobulin (preferably, IgG, most preferably, IgG1) as a dimeric domain. This is connected to the scFv fragment by means of a hinge region and linker region which also belong to IgG. Examples of such miniantibodies are described by Hu et al, 1996.

The term trispecific antibody (triabody) is used in the art to indicate a trivalent homogeneous trimeric scFv derivative (Kortt et al, 1997). Direct fusion of VH-VL would form a trimer without the use of linker sequences.

Fragments known in the art as miniantibodies having di-, tri-or tetravalent structures may also be derivatives of scFv fragments. Multiple polymerization can be achieved by means of a di-, tri-or quadruple coiled-coil structure (Pack et al, 1993 and 1995; Lovejoy et al, 1993).

The term "antibody fusion" or "antibody fusion protein" refers to a fusion/conjugate of a protein to an antibody or portion of an antibody. In particular, these include fusion proteins produced by genetic engineering, wherein a therapeutic protein is coupled to the Fc portion of an antibody to increase the half-life/stability of the protein in serum. The term also encompasses antibody fusions composed of peptides and antibodies or portions of antibodies.

For the purposes of the present invention, preferred host cell lines are hamster cells such as BHK21, BHKTK, CHO-K1, CHO-DUKX, CH-DUKX B1 and CHO-DG44 cells or derivatives/progeny of these cell lines. Particularly preferred are CHO-DG44, CHO-DUKX, CHO-K1 and BHK21 cells, in particular CHO-DG44 and CHO-DUKX cells. Also suitable are myeloma cells from mice, preferably NS0 and Sp2/0 cells and derivatives/progeny of these cell lines.

Examples of hamster and mouse cells that can be used according to the invention are given in table 1 below. However, derivatives and progeny of these cells, other mammalian cells, including but not limited to human, mouse, rat, monkey, rodent or eukaryotic cells (including but not limited to yeast, insect, avian and plant cells) may also be used as host cells for the production of biopharmaceutical proteins.

Table 1: production cell lines are known

Cell lines	Deposit number
		NSO	ECACC number 85110503
Sp2/0-Ag14	ATCC CRL-1581
		BHK21	ATCC CCL-10
BHK TK-	ECACC number 85011423
		HaK	ATCC CCL-15
2254-62.2 (BHK-21 derivatives)	ATCC CRL-8544
		CHO	ECACC number 8505302
CH-K1	ATCC CCL-61
		CHO-DUKX (=CHO duk-，CHO/dhfr-)	ATCC CRL-9096
CHO-DUKX B1	ATCC CRL-9010
		CHO-DG44	Urlaub et al, Cell 33[2 ]]，405-412，1983
CHO Pro-5	ATCC CRL-1781
		Lec13	(Stanley P. et al, 1984).
V79	ATCC CCC-93
		B14AF28-G3	ATCC CCL-14
HEK 293	ATCC CRL-1573
		COS-7	ATCC CRL-1651

U266	ATCC TIB-196
		HuNS1	ATCC CRL-8644
Per.C6	(Fallaux, F.J. et al, 1998)
		CHL	ECACC number 87111906

According to the present invention, the recombinant mammalian cell is preferably a rodent cell, most preferably a murine cell such as NS0 and especially a hamster cell such as CHO or BHK.

Host cells according to the invention are preferably fully established, adapted and cultured under serum-free conditions. Particularly preferably, the host cells are additionally fully established, adapted and cultivated in a medium which is not only serum-free but also free of any animal proteins/peptides.

Examples of suitable nutrient solutions include commercially available media, such as Ham's F12(Sigma, Deisenhofen, DE), RPMI-1640(Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), minimal essential Medium (MEM; Sigma), Iskoff's Modified Dulbecco's SMedium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA, USA), CHO-S (Invitrogen), serum-free CHO-Medium (Sigma), and protein-free CHO-Medium (Sigma).

The term "producer cell" or "producer cell (Produzenten-Zellen)" or "production clone" indicates a cell that can be used in a method for producing a protein. In particular, this includes genetically engineered cells that can be used for industrial production of recombinant proteins. In particular, within the scope of the present invention, the term includes expression of recombinant proteins and genetically engineered eukaryotic host cells for the production of such proteins. In particular, this includes monoclonal cell lines useful for the production of therapeutic proteins.

The present invention describes a method for cultivating single cells, which comprises the following steps: (a) incubating a population of cells, (b) isolating single cells from the population of cells and (c) incubating the single cells in a conditioned medium, i) the conditioned medium containing albumin, preferably Human Serum Albumin (HSA), secreted directly into the medium by the live cells of the trans-albumin gene. In one embodiment, the invention describes a method wherein albumin secreted by live cells transgenic for albumin has the same effect on recloning efficiency at significantly reduced concentrations as recombinant or purified albumin. In particular, the present invention describes a method for cultivating single cells comprising the steps of: (a) incubating a population of cells, (b) isolating single cells from the population of cells and (c) incubating the single cells in a conditioned medium i) which contains albumin (preferably Human Serum Albumin (HSA)) secreted directly into the medium by the live cells of the transalbumin gene, ii) the albumin secreted by the live cells of the transalbumin gene at a significantly reduced concentration to achieve the same effect on the efficiency of re-cloning as recombinant or purified albumin.

In one embodiment, the invention features a method in which albumin secreted by live cells transgenic for albumin has the same effect on recloning efficiency at a concentration reduced by at least two-fold as recombinant or purified albumin. In a preferred embodiment, the invention describes a method wherein albumin secreted by a live cell transgenic for albumin has the same effect on recloning efficiency at a concentration reduced by at least five fold as recombinant or purified albumin. In one embodiment, the invention describes a method wherein the albumin concentration in i) is reduced at least 2 fold (preferably, at least 5 fold) compared to the albumin concentration of a comparable culture mixture containing recombinant or purified albumin. In another embodiment, the invention features a method in which a single cell of steps (b) and (c) expresses a protein of interest.

In a preferred embodiment, step c) is carried out under serum-free conditions, preferably using chemically defined and/or insulin-free media. In one embodiment, the invention describes a process wherein in step (c) albumin is present at a concentration of less than 200mg/L, preferably less than 100mg/L, particularly preferably less than 60 mg/L. In another embodiment, the invention describes a process wherein albumin ii) in step (c) is present at a concentration that is 2 to 100 fold, 2 to 5 fold, 5 to 100 fold, 20 to 100 fold or 5 to 10 fold lower (preferably at least at a concentration that is 5 fold lower, most preferably at least at a concentration that is 10 fold lower).

In a preferred embodiment, the invention describes a method wherein in step (c) the cell supernatant of live cells of the transalbumin gene removed during days 1 to 4 of incubation is added. In a particularly preferred embodiment, the invention describes a process in which in step (C) the cell supernatant of the live cells of the transalbumin gene removed on day 2 of incubation is added.

In another embodiment, the invention features a method wherein the cell supernatant is taken from a cell culture having 1,000,000 to 4,000,000 (preferably, 1,000,000 to 2,000,000) cells per ml of the transalbumin gene. In a preferred embodiment of the invention, the cell serum is taken from a cell culture having 1,000,000 to 4,000,000 (preferably 1,000,000 to 2,000,000) cells per ml of trans-albumin gene and the albumin concentration in i) is reduced by at least 5 to 10 fold, preferably at least 10 fold, compared to the albumin concentration of a comparable culture mixture containing recombinant or purified albumin.

In another preferred embodiment, the invention describes a method wherein the conditioned medium in step c) is produced by adding live feeder cells transgenic for albumin. Preferably, up to 500000 transgenic feeder cells/ml are added. Also preferred is a process according to the invention wherein the albumin-transgenic feeder cells are retained in the culture medium of step c) for a period of at least 2 weeks. In a preferred embodiment of the invention, the conditioned medium of step c) is produced by the addition of live feeder cells of a trans-albumin gene, wherein 500000 feeder cells per ml of trans-albumin gene or less are added and the albumin concentration in i) is reduced at least 5-fold to 100-fold, preferably 20-fold to 100-fold (preferably also more than 100-fold) compared to the albumin concentration of a comparable culture mixture containing recombinant or purified albumin.

In another preferred embodiment, the invention describes a method wherein the conditioned medium in step (c) is produced by the fact that: the single cells of steps (b) and (c) are albumin transgenic cells. In a particularly preferred embodiment, the single cell is transgenic for albumin and the target gene and the single cell produces albumin and also the target protein. In one embodiment, the invention describes a method wherein in step c) a) live feeder cells transgenic for albumin or b) cell supernatant of live cells transgenic for albumin removed during the period from day 1 to day 4 of incubation, preferably day 2 of incubation, is added.

The invention also describes a cell produced according to a method of the invention.

The invention further describes a method for producing a protein of interest in cells, preferably in CHO cells, preferably under serum-free incubation conditions, comprising the following steps:

a) generating a population of cells containing a target gene encoding a target protein,

b) incubating the cells under incubation conditions that allow growth of the cells,

c) the single cells are isolated and stored in containers, e.g., in 96-well plates,

d) incubating the single cell in a conditioned medium in the presence of

a) Cell supernatants of live cells of the transalbumin genes taken during incubation days 1 and 3, preferably day 2, or

b) Live feeder cells transformed with albumin gene,

e) selecting cells corresponding to the expression level of the target protein,

f) harvesting the protein of interest, e.g., by separating cellular components from supernatant, and

g) purifying the target protein.

In one embodiment, the invention describes a method characterized in that the protein of interest is a recombinant protein, preferably a therapeutic protein, preferably an antibody or antibody fusion protein or antibody fragment.

In one embodiment, the invention features a method of characterizing a single cell line hamster or murine cell, preferably a mouse myeloma cell, more preferably a CHO or BHK cell or an NSO cell. In another embodiment, the invention features a method in which live cells transformed with an albumin gene are from the same species as the respective deposited cells. Preferably, the cell line is a "chinese hamster ovary" (CHO) cell. More preferably, the cell is a hamster or murine cell, preferably a mouse myeloma cell, particularly preferably a CHO or BHK cell or a NSO cell, depending on the species of the respective deposited cell.

In a particular embodiment, the invention features a method for autologous transgenic cells or feeder cells.

In another particular embodiment, the invention describes a method characterized in that the single-cell separation of step b) is carried out by "limiting dilution" or using "fluorescence activated cell sorting" (FACS) equipment.

In a preferred embodiment, the invention features a method for expressing a protein of interest in the cells characterized in steps (b) and (c). In particular, the protein of interest is a therapeutic protein, preferably an antibody, antibody fusion protein or antibody fragment.

The invention also describes a protein prepared by the method of the invention.

The invention further describes a method for selecting production cells using the method of the invention.

The invention further describes the use of the producer cells of the invention for the preparation of biopharmaceutical proteins.

The invention further describes a serum-free conditioned medium which makes it possible to cultivate single cells, comprising:

a) cell supernatants of live cells transformed with albumin gene obtained during the incubation period from day 1 to day 4, preferably at day 2, or

b) Live feeder cells for modulating the trans-albumin gene of a culture medium.

Preferably, the medium of the invention contains an albumin concentration of less than 200mg/L, more preferably less than 100mg/L, even more preferably less than 60 mg/L. Preferably, autologous feeder cells are used. In a preferred embodiment, the invention features a medium in which single cell line CHO cells are grown. Particularly preferably, the single cell expresses the protein of interest.

In order to put the invention into practice, conventional techniques generally known to those skilled in cell biology, molecular biology, cell culture, fermentation techniques, and the like are used, unless otherwise specified. The following experimental data are merely illustrative of the invention in nature and are not limiting.

Experimental part

Apparatus and method

Cultivation of cells

In cell culture flasksAt 37 deg.C in a humid atmosphere and 5% CO₂Cells CHO-DG44/dhfr-/- (Urlaub et al, 1983) were grown permanently as suspension cells in serum-free HyQ SFM 4CHO medium (HyClone) or BI autologous medium supplemented with hypoxanthine and thymidine. Cell count and viability were determined with a CEDEX cell counter (Innovatis, DE) or by trypan blue staining and subsequently at 1-3X 10⁵Cells were seeded at/mL concentration and every 2-3 days. Recombinant CHO-DG44 was used for single cell cloning. The cultivation of the cloned recombinant cells is carried out in a similar manner to these cells. In addition, hypoxanthine and thymidine free HyQ medium, EX-cell culture medium (JRH, USA) or BI's autologous medium can be used as the medium.

Can be cultured in a cell culture flask at 37 deg.C in a humid atmosphere and 5% CO₂NS0, which were used as suspension cells in serum-free hybridoma medium, animal component-free medium (Sigma, Aldrich, St. Louis, USA). Cell count and viability were determined with a CEDEX cell counter (Innovatis, DE) or by trypan blue staining and subsequently at 1-3X 10⁵Cells were seeded at/mL concentration and every 2-3 days.

Preparation of feeder cells by irradiation

Suspension CHO-cells (untransfected cells) grown in serum and protein free were centrifuged at 18O g for 10 min and the cell concentration was adjusted to 1X 10 in HBSS (Hank's balanced salt solution)⁶/mL。

The cells were then irradiated with a radioactive radiation source (Cs 137-irradiator, Gamma acell 2000, manufactured by Molsgaardmedical A/S, Denmark) at an energy delivery dose of 4 Gy/min. After irradiation, the cells were seeded at about 10000 cells/well in a 96-well microtiter plate in Ex-cell medium (JRH, USA) or BI autologous medium (e.g., TH-9) specific for the cells and at about 37 ℃ and 5% CO₂Stored in the incubation chamber atmosphere. HSA-producing feeder cells are produced in the same manner.

Thus, the method was carried out with NS0 cells, while the feeder cells were maintained/seeded in a specific medium specific for the cells.

Preparation of conditioned Medium

Conditioned media was obtained from the supernatant of the animal cell culture. The cells were placed at sufficient seeding density and incubated for 1-7 days. The cells and supernatant were separated by centrifugation and then sterile filtered. The obtained filtrate was used as a conditioned medium.

Determination of HSA concentration by ELISA

HSA concentrations were determined using the human albumin ELISA quantification kit manufactured by Bethyl (catalog No. E80-129) according to the manufacturer's instructions.

FACS-based cell storage (FACS-basierte zellablage)

Automatic cell storage (single cell or multiple cell storage) is carried out with the aid of a flow cytometer (Coulterepic Altra (Messrs. Beckman-Coulter, Miami, FL, USA) equipped with an argon laser (488nm) using an Autoclone unit, the cells in exponential growth phase are centrifuged off and absorbed in a buffer until the desired cell concentration is obtained, the cells are subsequently sorted using "Hypers Ortion" according to their position in the scattered light at a rate of 8000-.

During sorting of CHO cells, the cells are deposited in Ex-cell culture medium (JRH, USA) or BI autologous medium with the corresponding HSA supplement, and preferably IGF or insulin.

Therefore, for NS0 cells, cell storage was performed in hybridoma culture medium, animal component-free medium (Sigma, Aldrich, St. Louis, USA).

Calculating the efficiency of recloning

The recloning efficiency was calculated from the quotient of the number of positive wells per well plate and the total number of wells per well plate. Positive wells are defined as those in which exactly one clone is present.

Examples of the invention

Example 1: preparation of HSA-producing CHO-cells

CHO-DG44 cells grown in suspension in serum-free were transfected with plasmids carrying expression cassettes for Human Serum Albumin (HSA) and DHFR as selectable markers (Urlaub and Chasin, 1980). A population of stably transfected HSA-cells is produced by subsequent incubation in HT-free medium. A HSA-specific ELISA was used to select those cell populations that secrete most of HSA. These were subjected to gene amplification by incubation in the presence of Methotrexate (MTX). By subsequent limiting dilution, HSA-producing cell lines are prepared from the initial heterogeneous cell population and these cell lines are then tested for their specific HSA-productivity. These cell lines undergo substantially another round of gene amplification by cultivation in medium with high MTX concentration in order to further increase HSA secretion rate. Since MTX also slows down cell growth, the productivity improvement and growth characteristics of the obtained cell lines were investigated and the cell line with the highest productivity and good growth was selected. From this cell a cell bank was generated which could be used in all future experiments.

Example 2: increasing the efficiency of recloning with conditioned media from HSA-producing cells

One possible embodiment of the invention comprises the use of HSA-producing cells to increase the efficiency of recloning. Before recloning, the culture of HSA-producing cells is started, which medium is conditioned for several days, i.e.growth factors are released and, in particular, HSA secreted by the cells into the medium. On the day of recloning, the batch was removed by centrifugation, filtration (as appropriate) and mixing at 1: 1 ratio was added to the recloning medium and the conditioned medium was isolated from HSA-producing living cells.

To determine the optimal time course of medium conditioning, conditioned media were obtained from old cultures at day 2, day 3, day 4, day 5, day 6 and day 7 and used for recloning experiments. The positive control used was fresh medium to which 500mg/L of purified recombinant HSA had been added.

As can be seen from fig. 2A and 2B, the use of conditioned medium for HSA-producing cells significantly improved single cell storage efficiency compared to the negative control. By adding recombinant HSA (positive control), the recloning efficiency could also be increased, but not as well as by adjusting the medium for 2-5 days of HSA secreting cells. The HSA concentration of the conditioned medium continuously increased with time, but the recloning efficiency decreased when the medium was adjusted for 5 days or more. Surprisingly, the greatest efficiency improvement is obtained by means of conditioned medium on day 2, day 3 or day 4, with HSA concentrations (10mg/L to about 60mg/L) which are much lower than the concentration of recombinant HSA used in the positive control. This shows that secreted HSA is active even at low concentrations and has a 5-fold to 50-fold higher re-cloning efficiency enhancing efficacy compared to recombinant HSA.

Example 3: improvement of recloning efficiency in different media

To test whether the increase in recloning efficiency observed by using conditioned medium from HSA-producing cells could be operated independently of the medium, single cell recloning was performed in different media: in one batch, the monocytes were plated in BI autologous medium with CHO-DG44 feeder cells (FIG. 3A) and in another batch, the monocytes were plated in commercial medium without feeder cells (FIG. 3B).

In both media, the use of conditioned media from HSA secreting cells significantly improved the recloning efficiency, which was higher than the negative and positive controls (where recombinant HSA was added to the recloning medium). In batches without feeder cells (in commercial medium), no cells survived in the negative control, but 5-13% recloning efficiency was achieved by using conditioned medium of HSA-producing cells.

In both experiments, it was again shown that secreted HSA from the conditioned medium acted at significantly reduced concentrations and was therefore more efficient than HSA recombinantly added to the medium.

Example 4: increase in recloning efficiency by HSA-producing feeder cells

Conditioned medium from a primary culture of HSA-producing cells is added to the re-cloning medium before seeding the individual deposited cells. Therefore, HSA-containing medium was added to these cells only once at the beginning of the three-week budding period after recloning. In contrast, HSA-producing feeder cells can be continuously supplied with HSA to display single cell clones.

To investigate whether the use of HSA-producing feeder cells could further improve the recloning efficiency, CHO-DG44 cells were individually deposited in 4 96-well plates with HSA-producing and non-HSA-producing feeder cells. As in the previous example, the recloning medium contained 50% conditioned medium from the primary HSA-producing culture. After 3 weeks, the emerging monoclonal cell populations of each cell culture plate were counted and the recloning efficiency was calculated.

As shown in fig. 4, the recloning efficiency when using HSA-producing feeder cells was significantly higher than that of the well plates containing normal feeder cells. The increase is about 25%. Thus, a significant increase in the efficiency of re-cloning may alternatively be achieved by combining HSA-conditioned medium with HSA-producing feeder cells.

Example 5: HSA-producing host cells exhibit increased efficiency of re-cloning

Another possibility to increase the efficiency of recloning may be the use of HSA-producing host cells for the production of therapeutic proteins. In this way, during recloning, the individually deposited producer cells themselves can produce HSA and stimulate themselves by an autocrine loop. To examine whether this process actually improved the efficiency of re-cloning, normal CHO-DG44 cells and HSA-producing cells were seeded separately into DG 44-producing and HSA-producing feeder cells. After 3 weeks, the colonies were counted and the recloning efficiency was calculated for all three batches.

FIG. 5 shows that the efficiency of recloning was lowest in the dishes with unaltered CHO-DG44 host cells and feeder cells. On the other hand, if the HSA-producing cells are re-cloned, more monoclonal colonies will appear immediately. This shows that the probability of HSA producing cells to survive re-cloning and form a culture capable of replication is higher than that of non-secreting HSA cells.

The maximum re-cloning efficiency improvement is obtained by a combination in which HSA producing host cells are seeded in HSA producing feeder cell soil. The mixture further improves the recloning efficiency by more than 40 percent. Even an increase of more than 50% was obtained compared to the control mixture without HSA-producing feeder cells.

Example 6: re-cloning efficiency of NS 0-cells

Previous experiments have shown that the efficiency of re-cloning of hamster cells can be increased by means of secreted human serum albumin. These results may often indicate that HSA has a positive impact on the efficiency of recloning in each species.

To test this hypothesis, the therapeutic protein-producing murine NS0 hybridoma cells were recloned. The murine NS0 hybridoma cells were placed first in fresh medium and then in medium supplemented with conditioned medium from HSA-producing CHO cell cultures and then in a third batch containing HSA-producing feeder cells.

The use of HSA producing cells has a positive impact on the efficiency of recloning compared to recloning in fresh medium or conditioned medium of cell cultures without HSA secreting cells. This applies to the use of conditioned medium from HSA-secreting cells as well as HSA-producing feeder cells.

This shows that human HSA secreted by hamster cells may contribute to the re-cloning of murine cells.

Example 7: increasing the efficiency of re-cloning of human cells using secreted HSA

To validate this finding on a broader basis, human HEK 293-based producer cells were recloned in the presence and absence of conditioned medium from HSA-producing cells.

The use of HSA producing cells has a positive impact on the efficiency of recloning compared to recloning in fresh medium or conditioned medium of cell cultures without HSA secreting cells. This applies to the use of conditioned medium for HSA-secreting cells as well as HSA-producing feeder cells.

From this it can be concluded that: the secreted HSA has an influence on the recloning efficiency of the individually deposited cells of several species, i.e.all species in which it is active.

Example 8: re-cloning due to increased likelihood of recognizing highly productive cells by HSA-producing cells Efficiency of

The specific productivity levels of the producer cells of the heterogeneous cell population are normally distributed. This means that most of the cells have an average productivity, whereas few cells produce a large amount of recombinant product and only a small percentage produce a very large amount of recombinant product. This fact suggests that this effort is spent in cell screening and identifying high producing cells in the industrial cell line development program. It also means that the possibility of finding such high producers is directly related to the recloning efficiency: the more cells appear after recloning, with a greater probability of high producing cells being present.

To verify this conclusion in practice, the generation of secreted therapeutic proteins was performed by recloning a heterogeneous population of stably CHO-transfected cells. Some were placed in recloning medium (batch 1) to which 50% conditioned medium from parental CHO-DG44 cells had been added and some were placed in 50% conditioned medium (batch 2) from cultures of HSA-producing cells.

As in the previous example, more cells were grown into clonal cell lines in batches in which media from HSA-producing cells was used.

In the next step, 100 clones were selected from two re-cloning experiments and their productivity was determined by product-specific ELISA. The specific productivity distribution of 100 clones was shown to be normal in both cases. The mean of this curve clearly shifts to the right towards higher yields in 100 clones that appeared from cells re-cloned with medium from HSA-conditioned medium (batch 2). The average productivity values for the 10 highest producing cells from this batch 2 were higher than the average productivity values for the 10 highest producing cell lines of the re-clone batch (batch 1) without HSA producing cells. Thus, the cell line selected as the highest producing clone for the production of therapeutic proteins was from batch 2.

This confirms that more cells with higher yields are indeed obtained from a re-cloning batch with HSA-producing cells, thus increasing the likelihood of identifying them. At the same time, there is also a greater possibility to find a few cells that produce large amounts of recombinant protein product and thus make it possible to select and use high-producing clones for production.

Claims (25)

1. A method for culturing single cells comprising the steps of:

(a) culturing a population of cells, wherein the population of cells,

(b) isolating a single cell from the population of cells, and

(c) culturing the single cell in a conditioned medium containing albumin directly secreted into the medium by the live cells transgenic for albumin.

2. The method of claim 1, wherein the albumin is Human Serum Albumin (HSA).

3. The method of claim 1 or 2, wherein albumin is present in step (c) at a concentration of less than 200 mg/L.

4. The method of claim 3, wherein albumin is present in step (c) at a concentration of less than 100 mg/L.

5. The method of claim 4, wherein albumin is present in step (c) at a concentration of less than 60 mg/L.

6. The method of any one of claims 1 to 5, wherein in step (c) a cell supernatant of live cells transfected with albumin gene is added, which is taken during 1 to 4 days of culture.

7. The method of claim 6, wherein in step (c) a cell supernatant of live cells transfected with albumin gene is added, which is taken on day 2 of culture.

8. The method of claim 6 or 7, wherein the cell supernatant is taken from a cell culture having 1,000,000 to 4,000,000 cells per ml of transalbumin gene.

9. The method of claim 8, wherein the cell supernatant is taken from a cell culture having 1,000,000 to 2,000,000 cells per ml of trans-albumin gene.

10. The method of any one of claims 1-5, wherein the conditioned medium in step c) is produced by adding live feeder cells transgenic for albumin.

11. The method of claim 10, wherein up to 500000 transgenic feeder cells/ml are added.

12. The method of claim 10 or 11, wherein the transgenic feeder cells are continuously maintained in the medium of step c) for a period of at least 2 weeks.

13. The method of any one of claims 1-5, wherein in step (c) the conditioned medium is produced by the fact that the single cells of steps (b) and (c) are albumin-transgenic cells.

14. The method of claim 13, wherein in step c):

a) live feeder cells supplemented with a transgenic albumin gene, or

b) Cell supernatants of live cells transformed with albumin gene were added, which were taken from 1 to 4 days of culture.

15. The method of claim 14, wherein the cell supernatant of the live cells transgenic for albumin is taken from day 2 of culture.

16. A method of producing a protein of interest in a cell, comprising the steps of:

a) generating a population of cells containing a target gene encoding a target protein,

b) culturing the cell under culture conditions that allow the cell to grow,

c) the single cells were isolated and placed in a container,

d) culturing the single cell in a conditioned medium having

i) Cell supernatant of live cells transformed with albumin gene is taken out on 1 to 3 days of culture, or

ii) live feeder cells transformed with albumin gene,

e) selecting cells corresponding to the expression level of the target protein,

f) harvesting the target protein, and

g) purifying the target protein.

17. The method of claim 16, wherein cell supernatant of the live cells transgenic for albumin is removed on day 2 of culture.

18. The method of any one of claims 1 to 17, characterized in that the single cell is a hamster or murine cell.

19. The method of claim 18, characterized in that said single cell is a mouse myeloma cell.

20. The method of claim 18, characterized in that said single cell is a CHO or BHK cell or a NSO cell.

21. The method according to any one of claims 1-20, characterized in that said live cells transgenic for albumin are from the same species as said individual input cells.

22. The method of any one of claims 1-17, characterized in that said single cell in steps (b) and (c) expresses a protein of interest.

23. The method of claim 22, characterized in that the protein of interest is a therapeutic protein.

24. The method of claim 23, characterized in that the therapeutic protein is an antibody, an antibody fusion protein or an antibody fragment.

25. A serum-free conditioned medium capable of culturing single cells, comprising:

i. cell supernatants of live cells transferred with albumin gene taken during the culture on days 1 and 3, or

Live feeder cells for modulating the trans-albumin gene of the medium.