U.S. patent application number 09/766333 was filed with the patent office on 2001-07-26 for culture medium for insect cells.
Invention is credited to Haggstrom, Lena, Ohman, Lars.
Application Number | 20010009767 09/766333 |
Document ID | / |
Family ID | 10771979 |
Filed Date | 2001-07-26 |
United States Patent
Application |
20010009767 |
Kind Code |
A1 |
Ohman, Lars ; et
al. |
July 26, 2001 |
Culture medium for insect cells
Abstract
The present invention is directed to a culture medium for insect
cells, particularly for culturing insect cells for the expression
of proteins using a baculovirus expression system, characterized in
that the medium does not contain glutamine.
Inventors: |
Ohman, Lars; (Stockhom,
SE) ; Haggstrom, Lena; (Sollentuna, SE) |
Correspondence
Address: |
Docket Coordinator
WIGGIN & DANA
One Century Tower
New Haven
CT
06508-1832
US
|
Family ID: |
10771979 |
Appl. No.: |
09/766333 |
Filed: |
January 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09766333 |
Jan 19, 2001 |
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08913981 |
Dec 8, 1997 |
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6210966 |
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Current U.S.
Class: |
435/69.1 ;
435/348 |
Current CPC
Class: |
C12N 2500/05 20130101;
C12N 2500/32 20130101; C12N 5/0601 20130101; C12N 9/2471 20130101;
C12Y 302/01023 20130101 |
Class at
Publication: |
435/69.1 ;
435/348 |
International
Class: |
C12N 005/06; C12N
005/10; C12P 021/06 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 27, 1995 |
GB |
9506249.3 |
Mar 21, 1996 |
EP |
PCT/EP96/01281 |
Claims
1. An insect cell culture medium characterised in that the medium
does not contain glutamine.
2. An insect cell culture medium according to claim 1 which also
does not contain glutamate.
3. An insect cell culture medium according to claim 1 or 2 which
does not contain aspartate.
4. An insect cell culture medium according to any preceding claim
which contains an ammonium ion source.
5. A method of culturing insect cells comprising growing the cells
in a medium according to any preceding claim.
6. A method of obtaining a polypeptide including transforming
insect cells with recombinant baculovirus including a gene coding
for said polypeptide, and growing the transformed cells in a
culture medium that does not contain glutamine but which does
contain an ammonium ion source.
7. A method according to claim 5 or claim 6 wherein the cells are
from Spodoptera frugiperda.
8. A method according to claim 7 wherein the cells are from the
cell lines Sf-9 or Sf-21.
Description
[0001] This application concerns a culture medium for insect
cells.
[0002] The baculovirus expression system using insect cells has
become an important tool for production of recombinant proteins for
several reasons. First, the expression levels are often very high
compared to those obtained in other animal cells, such as
CHO-cells. Second, proteins produced using this system are often
biologically active due to the insect cells' ability to perform
post-translational modifications, folding and assembly of most
proteins. Third, the time from cloning to production of the protein
is short compared to the time needed to construct a stably
transformed animal cell line. Fourth, cell death inevitably follows
virus infection of insect cells. This can be an advantage over
other expression systems because it may permit better expression of
cytotoxic, regulatory or essential cellular genes.
[0003] Foreign proteins are produced during a lytic infection of
insect cells with a recombinant virus. Baculoviruses contain a very
late hyper-expressed gene, polyhedrin, which is not essential for
viral replication. Placing a gene under control of the polyhedrin
promoter allows the production of large quantities of a recombinant
protein. The strategy for production of a protein involves three
distinct stages:
[0004] (a) growing the insect cells from mid to late growth
phase;
[0005] (b) infecting the cells with virus containing a gene
encoding the protein of interest; and (c) harvesting and
purification of the protein product.
[0006] Insect cells are conventionally cultured in complex media
containing inorganic salts and sometimes organic salts, amino
acids, sugars, vitamins, trace elements, lipids and protein
hydrolysates. For example one commercially available medium,
called, IPL-41, is available from a number of suppliers. Apart from
these components, serum or yeast extract has conventionally been
added to provide undefined but essential nutrients. The amino acid
glutamine has hitherto been assumed to be essential for cultured
insect cells (Wang, M-Y et al Biotechnol. Prog. 1993, 9, 355-361;
Kamen, A. A. et al Biotecnol. Bioeng. 1991, 38, 619-628;Wang, M-Y
et al Biotechnol. Prog. 1993 in press), as it is for most cultured
mammalian cell lines.
[0007] We have surprisingly found that insect cells are capable of
synthesising glutamine themselves. The ability of insect cells to
grow without an external glutamine source has not been described in
the literature before. We have now shown that insect cells are,
indeed, able to grow without glutamine supplied to the medium and
that insect cells are, in fact, capable of growing in a medium
without glutamine, glutamate or aspartate.
[0008] According to one aspect of the invention there is provided a
medium, for example for culturing insect cells for producing
proteins or polypeptides using a baculovirus expression system,
characterized in that the medium does not contain glutamine.
[0009] The medium may also optionally not contain glutamate and/or
aspartate.
[0010] The medium of the invention can be used for growth of the
cells for the production of recombinant proteins.
[0011] The medium of the invention has several advantages over
traditional, glutamine-containing, insect cell culture media.
First, glutamine is generally considered problematic in cell
culture because of its instability. It decomposes spontaneously to
pyrrolidone carboxylic acid and ammonia in aqueous solution. This
instability is the major factor limiting the storage life of a cell
culture medium. A medium without glutamine thus results in a
markedly longer storage life. The medium of the invention is also
less expensive and simpler to prepare. Further, cells growing in
the medium of the invention exhibit a more efficient metabolism and
a decreased secretion of by-products that may be inhibitory to
growth.
[0012] Preferably, the medium comprises an ammonium salt or another
ammonium ion source or at least one amino acid or another compound
that can be converted to ammonium ions. An external ammonium ion
source is then used as a nitrogen source for biosynthesis of amino
acids. Preferably, the concentration of ammonium in the medium is
up to 20 mM. However, during certain conditions, as for example, in
substrate limited cultures, the cells may liberate ammonium
themselves which in turn can be used for biosynthesis. Under these
conditions addition of ammonium is not necessary for growth.
[0013] According to another aspect of the invention, there is
provided a method of culturing insect cells, the method comprising
growing the cells in a medium according to the invention.
[0014] According to a further aspect of the invention we provide a
method of obtaining a polypeptide including transforming insect
cells with recombinant baculovirus including a gene coding for said
polypeptide, and growing the transferred cells in a culture medium
that does not contain glutamine but which does contain an ammonium
ion source.
[0015] The insect cells may for example be from the fall armyworm,
Spodoptera frugiperda.
[0016] The preparation and use of a medium in accordance with the
invention will now be described, by way of example only, with
reference to the accompanying drawings FIGS. 1 to 4 in which:
[0017] FIG. 1 illustrates the growth of insect cells in different
media;
[0018] FIG. 2 illustrates the production of alanine and ammonium
ions by insect cells under different conditions;
[0019] FIG. 3 illustrates the growth of insect cells under
different conditions; and
[0020] FIG. 4 illustrates the growth of insect cells in different
media.
[0021] FIG. 5 illustrates the expression of the
.beta.-galactosidase gene from cells grown in a medium according to
the invention.
EXAMPLE 1
[0022] Spodoptera frugiperda (Sf-9) cells were cultivated in
suspension culture in spinner flasks (Techne, 125 ml) with 50 ml
medium, stirred at 60 rpm and incubated at 27.degree. C. A new
culture was started every second month from ampoules stored in
liquid nitrogen. The basic medium used (KBM502) had the following
composition in g l.sup.-1: glucose, 2.5; KCl, 2.87; CaCl.sub.2,
1.0: MgCl.sub.2, 1.07; MgSO.sub.4, 1.36; NaHCO.sub.3, 0.35;
NaH.sub.2PO.sub.4, 0.88; choline chloride, 0.02; glutamine 1.0;
L-arginine-HCl, 7.0; L-asparagine, 3.5; L-aspartic acid, 3.5;
L-glutamic acid, 6.0; L-glycine, 6.5; L-histidine-HCl, 2.0;
L-isoleucine, 0.5; L-leucine, 0.75; L-lysine-HCl, 6.25;
L-methionine, 0.5; L-phenylalanine, 1.5; L-proline, 3.5; L-serine,
5.5; L-threonine, 1.75; L-valine, 1.0; L-cystine-2 HCl, 0.5;
L-tryptophan, 1.0; L-tyrosine, 0.72; and, in .mu.g l.sup.-1;
para-amino benzoic acid, 320; biotin, 160; D-calcium pantothenate,
80; folic acid, 80; myo-inositol, 400; niacin, 160; pyridoxine-HCl,
400; riboflavin, 80; thiamine-HCl, 80; vitamin B-12, 240;
COCl.sub.2.6 H2O, 50; FeSO.sub.4.7H.sub.2O, 550; MnCl.sub.2O, 20;
(NH.sub.4).sub.6.(Mo.sub.7O.sub.244H.sub.2O), 40; CuCl.sub.2, 158;
and ZnCl.sub.2, 40. The medium was supplemented with 10% FCS
(Gibco), and 50 mg 1-1 Gentamicin (Sigma). In the bioreactor
experiments, 0.1% Pluronic F-68 (Sigma), 40 ppm Anti foam C (Sigma)
was added to the medium.
[0023] Glutamine, glutamate and aspartate were omitted from the
medium as indicated below. The concentration of NH.sub.4Cl (when
added) was 5 mM.
[0024] Spinner flask cultures
[0025] In the first experiment three media were tested a) KBM502
without glutamine, b) KBM502 with 5 mM NH.sub.4Cl but without
glutamine, and c) KBM502 with 1 g l.sup.-1 glutamine (control
culture).
[0026] In the second experiment the following media were tested a)
KBM502 with 5 mM NH.sub.4Cl, without glutamine, b) KBM502 with 5 mM
NH.sub.4 Cl, without glutamine or glutamate, c) KBM502 with 5 mM
NH.sub.4 Cl without glutamine, glutamate or aspartate.
[0027] Each medium was tested for growth of cells in triplicate.
Spinner flasks containing 50 ml of medium were inoculated to a cell
density of 2.5.times.10.sup.5 cells/ml.sup.-1. The cells were
counted once a day.
[0028] Bioreactor cultures
[0029] The medium used for bioreactor cultures was essentially the
same as KBM502, but with the following modifications: it contained
Tryptose Broth (Sigma) 2.6 g l.sup.-1; the amino acid composition
was slightly different (1.times.Grace's amino acid solution
modified for TC 100 (Sigma)) and the normal concentration of
glutamine was 0.6 g l.sup.-1.
[0030] Tank reactors with a working volume of 3.0 (Belach Bioteknik
AB, Stockholm, Sweden) equipped with one three blade marine
impeller (d=70 mm) were used. The stirring speed was 100 rpm and
DOT was controlled at 30% by intermittent sparging of oxygen at 200
ml min.sup.-1. Air was also passed above the surface at 100 ml
min.sup.-1. The pH of the medium was initially 6.2 but was not
controlled during culture. The culture temperature was maintained
at 27.degree. C. Inoculua were prepared from spinner flask
cultures. The cells were centrifuged and resuspended in fresh
medium prior to inoculation. The initial cell density in the
bioreactor was 2.times.10.sup.5 cells ml.sup.-1.
[0031] In batch cultures the medium was supplemented with 2.5 g
l.sup.-1 glucose and 0.6 g l.sup.-1 glutamine. In the glucose
fed-batch culture a glucose solution (15 g l.sup.-1) was
continuously pumped into the reactor(1.8 ml h.sup.-1) from the time
of exhaustion (42 h) of the amount of glucose supplied by the
tryptose broth; and 0.6 g l.sup.-1 of glutamine was supplemented
from the beginning of the culture. In the glutamine-limited culture
no glutamine was present in the initial medium but was fed (1.8 ml
h.sup.-1) into the bioreactor using a glutamine solution (3 g
l.sup.-1) from the beginning of the experiment. Glucose (2.5 g
l.sup.-1 was added from the beginning. The combined glucose and
glutamine fed-batch culture was fed initially with only a glutamine
solution but when the initial amount of glucose was exhausted (44.5
h), a mixed solution of glucose (15 g l.sup.-1) and glutamine (3 g
l.sup.-1) was fed at a rate of 1.8 ml h.sup.-1.
[0032] Sampling and analyses
[0033] Cell counting was performed using a Buker counting chamber
and cell viability was determined by trypan blue exclusion. Samples
for ammonium, lactate, glucose, amino acid and other organic acid
determinations were centrifuged and stored frozen until analyzed.
The ammonium ion concentration was measured with an ammonium
selective electrode (Orion). Lactate and uric acid were analyzed
enzymatically using test kits from Sigma while citrate and
succinate were determined by test kits from Boehringer Mannheim.
Glucose was analyzed with an YS1 glucose analyzer. Analyses of
amino acids were performed on a Biotronik LC 5001 amino acid
analyzer using ninhydrin as detection reagent.
[0034] Batch and Fed bath bioreactor cultures
[0035] The growth of Sf-9 cells and the concentration of glucose
and glutamine in a) a batch; b) a glutamine-limited fed batch c) a
glucose-limited fed batch; and d) a dual glucose and
glutamine-limited fed batch culture is shown in FIG. 1. Symbols:
viable cells, filled squares; glucose, open squares; glutamine,
open circles.
[0036] FIG. 2 illustrates the production of alanine and ammonium
ions by Sf-9 cells in a) batch culture, b) glutamine-limited fed
batch culture, c) glucose-limited fed batch culture, and d) dual
glucose- and glutamine-limited fed batch culture. Symbols: Alanine
--.smallcircle.-- and ammonium --.circle-solid.-- formation by Sf-9
cells.
[0037] In the batch culture the formation of alanine was
considerable--a typical metabolic response of insect cell cultures
(FIG. 2a). The concentration of alanine increased from 3 mM at the
start to almost 8 mM. On the contrary, the formation of ammonium
ions was insignificant.
[0038] In the glucose-limited fed batch culture, the glucose
feeding was started at 42 h when the initial quantity of glucose,
(unfortunately supplied by the tryptose broth) was consumed (FIG.
1c). After that the glucose concentration remained very low (below
0.2 mM) throughout the culture. The formation of alanine is not
only completely depressed, but alanine is actually consumed after
glucose limitation was established, while, at the same time,
ammonium production is triggered (FIG. 2c). The concentration of
ammonium ions increases abruptly from 0.5 mM to around 4 mM.
Ammonium formation continues, although at a lower rate, after
depletion of glutamine (150 h), indicating that it is released also
from other amino acids.
[0039] In the glutamine-limited culture, glutamine was fed at a
constant rate from the beginning resulting in a small increase in
concentration to a maximum of 0.5 mM whereafter it declined below
the detection limit (FIG. 1b). In this type of culture alanine
production takes place (FIG. 2b) but the accumulation is less than
that in the batch culture. No increase in ammonium ion
concentration is observed.
[0040] Finally, in the glucose and glutamine-limited culture, where
glutamine and glucose were fed simultaneously, low concentrations
of both substrates were established (FIG. 1d). Very little alanine
or ammonium was formed in this culture (FIG. 2d).
[0041] During glutamine limitation, the consumption of glutamine
was much less than that of any other amino acid as shown in Table
1.
[0042] This small amount seemed insufficient for the biosynthetic
need of the cells. Therefore it would appear that glutamine
biosynthesis occurs in the glutamine-limited cultures. The increase
in the concentration of glutamine in the stationary phase of the
batch culture, after exhaustion of the initial amount (FIG. 1a),
also supports the idea that Sf-9 cells can synthesise glutamine.
From Table 1 it is also evident that the consumption of glutamate
and aspartate increased in the fed batch cultures.
[0043] Growth of insect cells in medium without glutamine FIG. 3
illustrates the growth of cells cultivated in media with or without
glutamine and with or without 5 mM NH.sub.4Cl Cells cultivated in a
medium without glutamine but supplemented with NH.sub.4Cl, reached
almost the same maximum cell density, 2.9.times.10.sup.6 cells
ml.sup.-1, as cells cultivated in a standard medium containing
glutamine, although the growth rate is somewhat lower (FIG. 3).
This may be explained by the extra burden that the synthesis of
glutamine exerts on the cells. Cells grown in a medium lacking both
glutamine and NH.sub.4Cl grow very poorly; the maximum cell density
does not exceed 6.times.10.sup.4 cells/ml (FIG. 3). This experiment
has been repeated many times, and the cell culture has now been
maintained in a medium lacking glutamine for several months.
[0044] This experiment clearly demonstrates that Sf-9 cells are
capable of growing well without an external source of glutamine,
provided that ammonium ions are supplied to the medium. To add an
ammonium salt to a medium for animal cells has generally been
regarded as very unfavourable, as external ammonium is inhibitory
for most cultured animal cells, even at rather low levels. We
cannot see any negative effects of added NH.sub.4Cl below a
concentration of 20 mM.
[0045] Biosynthesis of glutamine, by glutamine synthetase, requires
the substrates glutamate and ammonium. We have shown earlier that
no ammonium was formed during growth of Sf-9 cells in a batch
culture with excess glucose (FIG. 2a) and in a glutamine limited
culture with excess glucose (FIG. 2b). Thus, external ammonium has
to be supplied as a nitrogen source for the biosynthesis of
glutamine. However, during glucose limitation Sf-9 cells excreted
ammonium into the medium (FIG. 2c). In such and similar cases it
may not be necessary to add ammonium to the medium.
[0046] Growth of insect cells in medium without glutamine,
glutamate and aspartate.
[0047] FIG. 4 illustrates the growth of insect cells in medium
without glutamine, glutamate and aspartate. FIG. 4 clearly shows
that Sf-9 cells are able to grow without external sources of
glutamine, glutamate, glutamate and aspartate, provided that
ammonium is supplied to the medium. No significant difference can
be seen in the growth between the three media.
[0048] These results indicate further that Sf-9 cells do indeed
synthesize glutamine, glutamate and aspartate because all three
amino acids are needed for various biosynthetic functions and for
protein synthesis. It is likely that glucose and ammonium ions are
the ultimate substrates for the synthesis of the three amino acids.
In this respect, the metabolism of Sf-9 cells resembles that of
many microorganisms, but it is a very uncommon type of metabolism
in cultured animal cells.
[0049] Further experimentation has confirmed that Sf-21 cells are
capable of growing in a medium containing no glutamine but to which
5 m M NH.sub.4Cl has been added.
EXAMPLE 2
[0050] Expression of .beta.-galactosidase in a glutamine-free
medium To study the effect of glutamine-free conditions on
recombinant protein production, Sf-9 cells were infected with a
recombinant baculovirus during growth in the glutamine-free but
ammonium supplemented medium (5 mM NH.sub.4Cl) and the result
compared to that from standard conditions (i.e. a medium with
glutamine but without added NH.sub.4Cl). The yield of
.beta.-galactosidase (24-72 h after infection, FIG. 5) indicates
that the production of the recombinant protein is not negatively
affected by the glutamine-free conditions.
1TABLE Consumption of amino acids in batch and fed batch (FB)
cultures of insect cells. Consumption (.mu.mol/10.sup.6
cells).sup.1 Glucose and Glucose Fed Glutamine Glutamine Amino acid
Batch Batch Fed Batch Fed Batch Glutamine 1.85 1.85 0.04 0.02
Glutamate 0.11 0.29 0.45 0.62 Aspartate 0.01 0.03 0.34 0.20
Asparagine 0.09 0.15 0.28 0.26 Serine 0.46 0.33 0.76 0.70 Lysine
0.13 0.22 0.37 0.40 Threonine 0.11 0.13 0.21 0.21 Arginine 0.23
0.27 0.40 0.43 Valine 0.14 0.11 0.20 0.27 Isoleucine 0.16 0.10 0.17
0.20 Leucine 0.29 0.25 0.36 0.36 Tyrosine 0.12 0.10 0.16 0.12
Phenylalanine 0.12 0.10 0.15 0.17 Tryptophan 0.04 0.07 0.08 0.07
Methionine 0.15 0.15 0.18 0.15
[0051] .sup.1Calculated from the beginning of the culture up to the
formation of two million cells/ml.
* * * * *