Note: Descriptions are shown in the official language in which they were submitted.
-1- 13l~`s~
METHOD OF INCREASING PRODUCT EXPRESSION
THROUGH SOLUTE STRESS
Field of the Invention
The present invention is in the qeneral
field of biochemical engineering. More specifically,
this invention is in the field of c011 and tissue cul-
ture dealing primarily with somatic hybrid cell cul-
ture.
Backqround of the Invention
With the advent of hybridoma technology and
the accompanying availability of monoclonal antibod-
ies, the application o-f such antibodies has escalated
into a variety of areas of the biological sciences.
For example, monoclonal antibodies have been used for
the study of cell surface antigens, for affini-ty puri-
fication of proteins, for histocompatibility testing,
for studying various viruses and for radioimmunoassay.
More recently, it has been recognized that monoclonal
an~ibodies may have medical application for drug tar-
geting and immunotherapy (C.H. Poynton and C.L.Reading, (1984) Exp Biol 44:13-33). With the in-
creased application of the antibodies in the biologi-
cal and medicinal sciences, there has come a con-
comitant demand for high levels of an-tibody produc-
tion.
To date, efforts have been undertaken todevelop culture condi-tions -to maximize cell culture
growth and thereby increase resultant product yield.
~ .
-2- 131 2 03 0
Early work in the development of chemically defined
animal cell culture media focused on the formulation
of such media to achieve rapid cell proliferation
~P.R. White, (1946) Growth 10-231-289, and C. Waymouth
(1974) J Natl Cancer Inst 53:1443-1448). Such media
incorporats specific nutrients, especially amino
acids, vitamins, purines, and pyrimidines. Today some
of the more widely used basal media for mammalian
cell cultures include Hams F-12, Dulbecco's modified
Eagle's medium (DM~), RPMI 1640, and Iscove's modified
DME. All of these above-referenced basal media are
also supplemented with several trace metals and salts,
including the major cations (potassium, sodium, cal-
cium, magnesium and the like) with concentration val-
ues near isotonic levels. The role of inorganic nu-
trition in cell culture is discussed in a number of
references including R.A. Shooter and G.O. Gey, (1952)
Br J Exp Pathol 33:98-103; C. Waymouth, (1974) supra;
J.R. Burch and S.J. Pert, (1971) J Cell Sci 8:693-700;
~.G. Ham, Growth of Cells in Hormonally Defined Media,
Cold Spring Harbor Conferences on Cell Proliferation,
Vol. 9, Sato, Pardee and Sirbashin, eds., 1982.
Culture media have been developed spe-
cifically for low serum and serum-free mammalian cell
cultures for production of monoclonal antibodies. One
such serum-free medium is disclosed in European Patent
Publication 076,647, published 13 April 1983. Other
media have been developed by changing levels of sup-
plements such as trace elements, vitamin and hormone
additives wherein variations in the traditional basal
media are slight. References to such media include,
for example, D. Barnes and G. Sato, (1980) Cell
_:649-655; W.L. Cleveland et al (1983) J Immunol
Meth 56:221-234; N. Iscove and F. Melchers, (1978) J
Exp Med 147:923-933; T. Kawamo-to et al (1983) Anal
Biochem 130:445-453; J. Kovar and F. Franek, (1984)
_3_ l 31 20~0
Immunol Lett 7:339-345; H. Murakami et al (1983)
Agric Biol Chem 47(8):1835-1840; H. Murakami et al
(1982) Proc Natl Acad Sci USA 79:1158-1162; H. Muzik
et al (1982) In Vitro 18:515-524; and S.D. Wolpe, ~'In
Vitro Immunization and Growth of Hybridomas in Serum-
Free Medium", in J.P. Mather, ed., "Mammalian Cell
Culture," Plenum Press, New York, 1984.
In addition to providing the right kinds and
amounts of nutrients, the culture medium must also
provide suitable physiochemical conditions. Param-
eters that are important for clonal growth of hybrid-
oma cell culture include osmolarity, pH buffering,
carbon dioxide tension, and partial pressure of
oxygen. These all must be adjusted to optimal values
for multiplication of each type of cell with, prefer-
ably, minimal or no amounts of serum and minimal
amounts of protein. Other physical factors such as
temperature and illumination must also be controlled
carefully.
Effor-~s to increase antibody yield have
focused primarily on means to optimize cell growth and
cell density. The optimal conditions for cell growth
of ma-mmalian cell culture are generally within narrow
ranges for each of the parameters discussed above.
For example, typical culture conditions for mammalian
hybridoma cell culture use a basal culture medium sup-
plemented with nutritional additives, pH in the range
of 6.8 to 7.4 at 35-37C.
As a general point of reference, antibody
titers from murine hybridoma cell lines are highly
variable from cell line to cell line and range
typically from 10 to 350 ug/ml (K.J. Lambert et al
(1987) Dev Indust Microbiol 27:101-106). Human mono-
clonal antibody expression from human/human or human/
mouse fusions are also highly variable from cell line
to cell line and range typically from 0.1 to 25 ug/ml
1 3 1 203~
4- ;
(R. Hubbard, "Topics in Enzyme and Fermentation
Biotechnology," chap. 7, pp. 196-263, A. Wiseman, ed.,
John Wiley & Sons, New York, 1983). These values are
indicative of culture conditions that are op-timized
for cell growth and cell viability.
Another example from the literature
documents ~hat, at least for some cell lines, mono-
clonal antibody production proceeds even after a cul-
ture stops growin~ (D. Velez et al, (1986) J Imm
Methods 86:45-52; S. Reuveny et al, (1986) ibid at p.
53-59). Thus, one strategy for increasing monoclonal
antibody yield has been to develop culture conditions
that allow growth of hybridomas to higher cell densi-
ties and to recover the antibodies late in the sta-
tionary phase of cell culture. W. Arathoon and J.Birch, (1986) Science 232:1390-1395 reported that a
1,000 liter hybridoma fermentation produced about 80
grams of monoclonal antibody during the growth phase
and another 170 grams of antibody during an extended
stationary/death phase. It is not known the means, if
any, by ~hich -the stationary phase of growth was ex-
tended.
Another approach from the literature to in-
creasing antibody production is to achieve high cell
densities by cell recycle o-r entrapment methods.
Examples of these me-thods include ho]low fiber re-
actors (G.L. Altshuler et al (1986) Biotechnol Bioenq
XXVIII, 646-658); static maintenance reactors (J.
Feder et al, Canadian Paten-t 1,210,352 issued 26 Au~. 1986;
ceramic matrix reactors (A. Marcipar et al (1983)
_ nals N.Y. Acad Sci 413:416-420); bead immobili~ed
reactors (K. Nilsson et al (1983) Nature 302:629-630);
perfusion reactors (J. Feder and W.R. Tolbert, (1985)
Amer Biotechnol Lab III:24-36) an~ others. In some
cases, a res-ting" cell culture s-tate is reported to
be achieved by reducing levels of nu-trien-ts in the
~. " .
.
1 3 1 2030
--5--
medium (as by reducing serum or protein supplement
levels) with antibody production continuing while
growth is slowed.
While a variety of methods to increase anti-
body yield from hybridoma cell culture are beingexplored, the primary focus is still on the optimiza-
tion of cell growth. We have discovered that culture
conditions for growth optimization and for optimal
product expression may differ and that product expres-
sion can be increased under condi-tions of solu-te
stress, created by the addition of certain solutes,
notwithstanding the resulting growth inhibitory
effects.
The concep-t of subjecting animal cells,
especially mammalian cell cultures, to an environment
of solute stress to produce higher product expression
yields, such as increased antibody titers, has not
been reported. One means for introducing such an en-
vironment to the culture is through salt addition
which is easily monitored by measuring the osmolarity
of the culture medium.
Media osmolarity for mammalian cell culture
is usually held in the range of 280-300 mOsM/kg (W.B.
Jakoby and I.~. Pas-tan, Meth Fnzymol, vol. LVIII,
25 "Cell Culture", Academic Press (1979), pp. 136-137).
Of course, the optimal value may depend upon the spe-
cific cell type. For example, as reported in "Tissue
Culture, Methods and Applications", edited by P.F.
Kruse, Jr., and M.K. Patterson, Jr., ~cademic Press
30 (1973) p. 704, human lymphocytes survive best a-t low
(about 230 mOsM), and granulocytes at higher osmolar-
ities (about 330 mOsM). Mouse and rabbit eggs develop
optimally in vivo at around 270 mOsM, 250-280 mOsM
being satlsfactory, while above 280 mOsM development
is retarded. Iscove reports 280 mOsM to be optimum
for grow-th of murine lymphocytes and hema-topoietic
1 -~ 1 20 30
--6--
cells, and Iscoves modified DME is adjusted for this
growth promo~ing osmolarity (N.N. Iscove, ~1984)
"Method for Serum-Free Cul-ture of Neuronal and
Lymphoid Cells," pp. 169-185, Alan R. Liss, ed., New
York.
The spread of quality control osmolarity
values on a number of commercially available tissue
culture media is provided in a table beginnin~ at page
706 in the "Tissue Culture, Methods and Applica-tionsl'
reference, supra. The osmolarity values given therein
reflect the 280-300 mOsM/kg range used for mammalian
cell culture.
Ano-ther means to introduce an environment of
solute stress in the cell culture is through the addi-
tion of cellular metabolic products, such as lacticacid and ammonia. These products are generally known
to be growth inhibitory agents and strategies to
reduce the level of these products in the culture
medium in or~er to enhance cell growth have been
reported. T. Imamura et al (1982) Anal Biochem
124:353-358; A. Leibovitz, (1963) Am J Hyq 78:173-
180; S. Reuveny et al (1986) J Immunol Meth 86:53-59;
J.S. Thorpe et al (1987) "The Effect of Waste Products
of Cellular Metabolism on Growth and Protein Synthesis
in a Mouse Hybridoma Cell Line", Paper #147 presented
at American Chemical Society National Meeting, Aug.
30-Sept. 5, 1987, New Orleans, La.--Symposium on
Nutrition and Metabolic Regulation in Animal Cell
Culture Scale-Up; and M.W. Glacken et al (1986)
_iotech & Bioeng XXVIII:1376-1389.
Contrary to the -teaching in the art which
cautions against major adjustments to culture media
osmolarity and other physiochemical parameters, we
have found that introducing an environment of solute
stress during fermentation can favor an increase in
specific (per cell) antibody expression and/or in-
~ 7 t ~ 1 2030
creased culture longevity which can result in an increase inantibody titer. It is to such a concept that this invention
is directed. Briefly, in a preferred embodiment of the
invention, an approach to mammalian cell culture which
further optimizes yield of antibody production has been
developed in which hybridoma cells are cultured uncler
conditions of controlled solute stress. Optionally, the
method incorporates prior art advances including the culture
of hybrid mammalian cell lines in serum-free media or in
high density culture to reduce costs and facilitate
purification.
Summary of the Invention
Therefore, this invention is directed to a method
for increasing expression of a protein in a mammalian cell
culture, which already provides for all cell growth
requirements, and recovering the protein from the culture,
the expression i5 increased above the expression level at
optimal growth, comprising adding a solute to the cell
medium at a level above that for optimal cell growth to
create stress on the cell, as expressed by an inhibitory
effect on cell growth or cell densiky; and recovering and
purifying the protein from the cell culture.
In another aspect of this invention is provided a
method to determine the solute level in a cell culture
medium ko produce the highest protein product expression
from a cell, the culture medium has all the requirements
necessary for optimal growth comprising; determining the
concentrations of solutes necessary for optimal cell growth;
increasing the concentrations of one or more solutes to
place the cell under stress; and determining the
concentrations which produce more protein product.
.
- 8 - l 3 1 203 0
A preferred method of this invention comprises
culturing human IgM-producing hybridoma cells and another
preferred method comprises culturiny hybridoma cells which
produce IgG.
These and other objects of the invention will be
apparent from the following description and claims. Other
embodiments of the invention embodying the same or
equivalent principles may be used and substitutions may be
made as desired by those skilled in the art without
departing from the present invention and the purview o~ the
appended claims.
Brief Description of the Drawinqs
Figure l shows the effect of 400 mOsM media on
antibody yields of human/human/murine trioma D-234 cells in
serum-free HL-1 media. The closed circles represent cell
growth in 300 mOsM media and the open circles represent the
resulting IgM antibody yield. The closed squares represent
cell growth in 400 mOsM media and the open squares represent
resulting IgM antibody yield.
Figure 2 shows the effect of ammonium chloride on
production of antibodies of D-234 cells. The closed circles
represent cell growth in the absence of ammonium chloride
and the open circles represent the resulting IgM antibody
yield. The open triangles represent cell growth in the
presence of 10 mM ammonium chloride and the closed triangles
represent resulting antibody yield.
DescriPtion of the Preferred Embodiments
As used herein the term "hybridoma" refers to a hybrid cell
line produced by the fusion of two or more cell lines to
produce an immortal cell line producing a desired product
- 8a - 1 31 203D
(such as an antibody). The term includes hybrids produced
by the fusion of a myeloma cell line and an antibody
producing cell (such~as a splenocyte or plasma cell). The
term also includes prog-
:, f.
1 3 I 203~)
g
eny of heterohybrid myeloma fusions (the result of a
fusion with human B cells and a murine myeloma cell
line) subsequently fused with a plasma cell, referred
to in the art as trioma cell lines.
5As used herein the term "animal" refers -to
any mammalian, insect or invertebrate species.
"Mammalian" indicates any mammalian species,
and includes rabbits, mice, dogs, cats, primates and
humans, preferably humans.
10As used herein the term "solute" indicates a
water soluble agent, including but not limited to in-
organic salts and the correspon~ing ions thereof;
~` o~ganic polyols, including~g~c~ro~ ~ d sugars such
'' as, for example, glucose, mannose, fructose and
mannitol; and metabolic products such as, for ex-
ample, lactate or ammonia; which is effective in pro-
ducing increase~ product expression.
As used herein -the term "solute stress~
refers to the addition of solutes in such concentra-
tions, a-t least above that concentration determined
for optimal cell growth, that produce a growth in-
hibitory effect or reduced final cell density, that
is, a growth rate or maximum cell density less than
that determined for optimal growth. ~owever, the
level of product expressed at this reduced growth
level is comparatively greater than that level of ex-
pression achieved at the optimal growth rate owing to
an increase in specific (per cell) product expression
rate or an increase in longevity of the culture.
30As used herein the term "osmolality'l refers
to the total osmotic activity contributed by ions and
nonionized molecules to a media solution. Osmolality,
like molality, relates to weight of solvent (mOsM/kg
H2O) while osmolarity, like molarity, relates to vol-
ume (mOsM/liter solution). Osmolality is one method
used to moni-tor solute stress. St~ndard osmolality
-10- 1 Jl 2~0
refers to the optimum range of clonal growth of mam-
malian cells which occurs at 290-~30 mOsM/kg.
In a preferred embodiment of the invention,
- methods have been developed for the high~level produc-
tion of mammalian, preferably human~ monoclonal anti-
bodies for use as diagnostic reagents or for use in
human therapy. In particular, a method of determining
the op-timal level of product expression in mammalian
cell culture has been developed wherein the concentra-
tion of a solute of interest in a culture mediwn com-
position for optimal product expression is different
than the culture medium cornposition determined for
optimal cell growth, which method comprises:
a) growing the mammalian cell culture in
medium to determine optimal cell growth;
b) varying the concentration of -the solute
in the culture medium to a concentration above that
optimal for cell growth which concentration is effec-
tive to create an environment of solute stress on the
cell culture;
c) monitoring the product expression under
the varying solute concentrations to determine op-timal
product expression; and
d) selecting the solute concentration that
provides the optimal combination of cell growth and
product expression which allows for optimal productiv-
ity.
Following the methodology set forth herein,
one is able to determine -the solute concentration that
provides the optimal combination of cell growth and
product expression for any particular cell line of
interest. Once -the solute concentration has been
determined, one is able to create an environmen-t of
controlled solute stress for culturi.ng the mammalian
cell lines and thereby s-timulate specific (per cell)
product expression and/or increase culture longevity,
I 3 1 ~030
notwithstanding the inhibitory growth effect on the
cul-tured cells.
The mammalian cell culture used in the
present invention includes, but is not limited to, any
of a number of cell lines of bo-th B-cell and T-cell
origin including murine thymic lymphoma cells, human
myeloma cell lines, and human lymphoblastoid cells and
hybridomas. Accordingly, the product -to be op-timized
includes growth factors, lymphokines, and monoclonal
antibodies. The cell cultures may include cell lines
which are found to naturally produce such desired pro-
ducts, or have been manipula-ted by genetic engineering
techniques to produce recombinant products.
Solute stress is introduced into the cell
culture fermentation by the addition of one or more
solutes which effectively inhibit optimal cell growth.
The solute can be added at various time periods during
the fermentation including prior to, during or aEter
the addition of cells. While such changes to the cul-
ture media negatively affect the growth of culturedcells (given the narrow growth parameters known for
optimal cell growth) the present invention lies in the
discovery that culturing cells in such an environment
of solute stress can positively impact specific cell
productivi.ty and culture longevity, thereby increasing
product yield.
Solute stress which is effective in increas-
ing the product yield can be achieved by increasing
the concentrati.on of a solute already presen-t in a
culture medium or introducing a new solute to the
medium.
In the method of the invention, a sub-lethal
solute concentration range is firs-t determined in
order to study the solute inhibi-tory growth effect.
This determination is necessary as each cell line may
have unique tolerance levels to the selected solute.
- 12 - 1 3 ~ 2 0 ~ O
As a second step, various sub-lethal concentrations are
studied in more detail to establish the conditions for
optimal cell productivity which is responsible for increased
product expression. From the data thus generated, one may
determine the solute concentration that provides for the
optimal combination of cell growth and product expression.
The following discussion, concerning the various
types of solutes that may be used in the methods of the
present invention, also provides a number of preferred
concentration ranges that have been determined for specific
hybridoma cell lines. Other cell lines may have somewhat
different tolerance levels. These ranges are provided as a
guide for determining the optimal combination of growth and
product expression levels for a variety of cultured cells and
are not to be construed as a limitation of the invention.
The concentration ranges provided herein are a good indicator
of a possible concentration range for the specific cell line
of interest.
The solutes of the invention comprise a number of
inorganic salts and ions thereof, including, for example,
sodium chloride, potassium chloride, calcium chloride,
magnesium chloride and ~he like, and combinations thereof.
Preferred salts include sodium chloride and combinations of
sodium chloride and potassium chloride. An effective
concentration range for the increased production of
monoclonal antibodies by the cell lines D-234 and T-88, using
salts such as sodium chloride is 340 to 460 mOsM/kg, with 350
to 400 mOsM/kg being more preferred for the cell line D-234
and 400 to 450 mOsM/kg being more preferred for the cell line
T-88. An effective concentration for the increased per cell
productivity of monoclonal antibodies by the cell line
454A12, using sodium chloride, is about 400 mOsmol/kg.
The concentration values given above, as well as
all concentration ranges provided herein regardless of the
method of solute concentration meas-
-13- 1 31 2030
urement used, have been established prior to the
addition of cells. However, the solute may be added
before, duxing or after cell addition. The timing of
the solute addition is generally not critical, as it
has been found that increasing solute stress by, for
example, salt addition, may be performed at various
time points during the exponential phase of the growth
cycle to achieve an increase in antibody yield. Of
course, one skilled in the art will appreciate that
the concentration of the metabolic solutes will
increase during the course of -the fermentation.
In addition to the aforementioned salts, it
has been found that solutes which are generally be-
lieved to have inhibitory growth effects may also be
used in the present invention. For example, lactic
acid, a major metabolic end product of glycolysis in
hybridoma cell culture, participa-tes in the lowering
of the pH during growth, producing sub-optimal growth
conditions. The lactate ion itself, may also be
growth inhibitory. Efforts have been made to reduce
lactic acid production by replacing glucose with
alternative sugars (i.e., fructose and galactose) that
are less easily metabolized to lactate. It has been
assumed that reduction of the level of lactate in the
culture medium would enhance both cell growth and
antibody production.
However, the present invention demonstrates
that the presence of lactate during fermentation can
effectively increase antibody yield notwithstanding
its inhibitory growth effec-ts. Using the methodology
of the present invention, a sub-lethal concentration
range (0 to 100 mM sodium lactate) was first deter-
mined in order to study the lactate inhibition effect.
Various sub-lethal concentrations of sodium lactate
are subsequently tested for the effect on produc-t
- 14 13~2030
expression. For the cell line D-234, an effective
concentration range for sodium lactate is 40 to 60mM.
Ammonia is another substance that has concerned
cell culturists due to its negative effects on cell growth.
It is produced by cellular metabolism of amino acids as well
as by spontaneous decomposition of glutamine. It has been
assumed that reduction of ammonia in hybridoma cultures would
banefit both cell growth and antibody production. However,
as demonstrated herein, an increase in antibody titer was
observed despite the inhibition of cell growth in the
presence of ammonium chloride. For the cell line D-234, a
preferred concentration range for ammonia chloride addition
is 3 to 20mM, with 10-15 mM being more preferred.
The organic polyols useful in the invention include
glycerol, polypropylene glycol and a variety of low molecular
weight sugars including, for example, glucose mannose,
fructose and mannitol. Of these organic polyols, glucose is
preferred, and for the cell line D-234, an effective
concentration range for glucose is 6 to 20 g/l, with 7 to 15
g/l being preferred. Another preferred organic polyol is
polypropylene glycol. For the cell line 454A12, an effective
concentration of polypropylene glycol is about 8~1/L.
Ths method of the invention is operable with any of
a variety of well-known and/or commercially available
mammalian cell culture media. Such suitable culture media
includes serum-free media such as HL-l (Ventrex Labs,
Portland, ME), HB104 (Hana Biologicals, Berkeley, CA),
Iscove's DME medium (Gibco, Grand Island, NY) and RPMI-1640
medium (Gibco) or media supplemented with serum. The
hybridomas used in the present method are preferably adapted
for growth and maintenance in serum-free medium for
large-scale, reproducible spinner culture production of
monoclonal antibodies using, for example, a step-wise method.
The method of the invention has been shown to
increase antibody titer regardless of the presence
~, ;,
~ 15 - 1 3 1 2030
or absence of serum in the medium. The cell lines used in
the present invention may be cell lines of diverse mammalian
origin. Rat, mouse and human embodiments are contemplated,
with human embodiments illllstrated in the examples which
follow. The antibodies may be of any class, including IgG
and IgM, with IgM and IgG types being specifically
exemplified herein. The human embodiments are the products
of triomas synthesized by somatic cell hybridization using a
mouse x human parent hybrid cell line and Epstein-Barr virus
(EBV)-transformed human peripheral blood lymphocytes (PBLs)
or splenocytes from non-immunized volunteers or volunteers
immunized with available Gram-negative bacterial vaccines or
inactivated Gram-negative bacteria. Fresh PBLs or
splenocytes (not transformed) may be used, if desired~ A
detailed description of the synthesis of the hybridomas,
including the fusion protocol, ELISAs and hybrid screening
procedure exemplified in the following examples is disclosed
in Canadian Application Serial No. 578,020.
8riefly, the mouse-human heterohybrid fusion
partner designated F3B6 was constructed by fusing human PBL B
cells obtained from a blood bank with the murine plasmacytoma
cell line NSl obtained from the American ~ype Culture
Collection (ATCC) under ATCC No. TIB18 (P3/NSl/1-AG4-1). The
resulting hybrid cells were adapted for growth in 99%
serum-free medium and deposited with the ATCC under ATCC No.
HB-8785.
The heterohybrid F3B6 cells and positive
EBV-transformed PBL B cells were then used to construct
hybridoma cells lines which secrete antibodies illustrative
for use in the method of the present invention. A preferred
strategy for preparing and identifying such hybrids follows~
Cells (PBLs, splenocytes, etc.) are panned on cell-wall
lipopolysaccharide (LPS) (an endotoxin of a gram-negative
bacteria which produces bacteremia) coated tissue culture
plates, the EBV transformed and fused to the tumor fusion
partner (mouse myeloma x human s cell or rate myeloma).
Panning involves incubation of the popula-
1 3 1 2030
-16-
tion of immunocompetent cells on a plastic surface
coated with the relevant antigen. Antigen-specific
cells adhere.
Following removal of non-adherent cells, a
population of cells specifically enriched for the
antigen used is obtained. These cells are transformed
by EBV and cultured at 103 cells per microtiter well
using an irradiated lymphoblastoid feeder cell layer.
Superna-tants from the resulting lymphoblastoid cells
are screened by ELISA against an E. coli Rc LPS and a
Salmonella Re LPS. Cells that are positive for
either Rc or Re lipid A LPS are expanded and fused to
a 6-thioguanine-resistant mouse x human B cell fusion
partner. If the mouse x human B cell fusion partner
is used, hybrids are selected in ouabain and aza-
serine. Supernatants from the Rc or Re positive
hybrids are assayed by ELISA against a spectrum of
Gram-negative bacteria and purified Gram-negative bac-
terial LPSs. Cultures exhibiting a wide range of
activity are chosen for in vivo LPS neutralizing
activity. Many but not all antibodies so produced are
of the IgM class and most demonstrate binding to a
wide range of purified lipid A's or rough LPS's. The
antibodies demonstrate binding to various smooth LPS's
~5 and to a range of clinical bacterial isolates by
ELISA.
Two of the hybridoma cell lines which
produce the Gram-negative bacteri.al endotoxin blocking
antibodies described above were used to illustrate the
methods of the present invention. D-234 and T-88 are
representative of hybridomas used in the methods of
the present invention to produce increased yields of
their respective monoclonal antibodies. D-234 was
adapted to growth and maintenance in serum-free medium
for large-scale production of monoclonal antibodies.
The D-234 hybridoma was crea-ted from a fusion of the
1 3 1 L 0 3 (~
heterohybrid fusion partner F3B6 and human B lympho-
cytes; a hybridoma sample adapted for growth in
serum-free media was deposited with the ATCC under ac-
cession number HB-8598. The T-88 hybridoma is a
fusion product of the same heterohybrid F3B6 and human
splenocytes from a lymphoma patient. ~ sample of this
hybridoma (that was not adapted for growth in serum-
free media) was deposited with the ATCC under acces-
sion number Hs-9431. In addition, a subsequent
hybridoma passage of D-234 was deposited with the
ATCC under accession number HB-9543. These latter two
hybridoma cell lines are specifically exemplified in
the following examples.
The murine-murine hybridoma cell line, 454A12,
used as an example here was formed from the fusion of a
mouse splenocy~e and a mouse myeloma fusion cell partner.
This hybridoma produces IgG monoclonal antibodies specific
for human transferrin receptor. The 454A12 hybridoma, its
production, and the antibody it produced were described in
U.S. patent application, Serial No. 069,867, "Anti-human
Ovarian Cancer Immunotoxins and Methods of Use Thereof",
filed July 6, 1987, applicants Bjorn, M.J. et al.
Examples
The following examples are illustrative of
this invention. They are not intended to be limiting
upon the scope thereof.
Example 1
Culture of D-234
A one ml ampoule of frozen D-234 stock (ATCC
HB-9543) was thawed quickly in a 37 C water bath. The
contents were aseptically added to 100 ml prewarmed,
pregassed, serum-free HL-l medium (Ventrex Labs,
Portland, Me) supplemented with 0.1% Pluronic-~ polyol
S
i .
-17a- 1 31 2330
F-68 and 8 mM L-glutamine in a 250 ml Erlenmyer flas~
with a loosely fitted plastic screw cap. The flask
was placed in a humidified incubator (36.5C, 90%
relative humidity and 5% CO2) and cultured with
shaking at 100-120 rpm.
This parent culture was subcultured during
mid-exponential phase, about 2-4 days after inocula-
tion, when the cell density was approximately 5 x 105
to 1 x 106 viable cells per ml. The subcultures were
grown in the daughter flasks under the same culture
~.
;
1312~30
-18-
conditions as above, starting with the initial
inoculum of 1 x 105 and 5 x 104 viable cells/ml. The
cells were counted using a Coulter Counter, and
viability was determined by trypan blue exclusion
using an hemocytometer. Maximum total cell densities
were around 1.7 million with viable cell densities
around 1 million.
For standard batch production, the cultures
were allowed to grow to completion which occurs about
7 to 10 days irom planting by which time cell
viability had declined to 30~ or less. The cells were
harvested by centrifugation (3,000 rpm for 5 min) to
separate the cells and purify the antibodies.
The resulting antibody yield was determined
by enzyme-linked immunoadsorbent assay (ELISA) using a
standard IgM ELISA but modified by using a high salt
(i.e., at least 0.5 M NaCl) assay buffer. IgM titers
were around 40 ug/ml.
Example 2
Effect of Salt Addition on IqM Production In D-234
The following treatments were set up in 100
ml working volume shake flasks at standard planting
C~ densities in HL-1 with 0.1% Pluronic~ ~-68 and 8 mM
glutamine. ~ 3.75 M salt solution (27:1 molar ratio
NaCl:KCl) was used to increase salt concentration
beyond that of the standard HL-l medium.
Approximately 1 x 105 viable cells/ml were
used to inoculate the aforementioned culture medium,
which was used as the control sample. In addition, 1
x 105 viable cells/ml were inoculated into a ~00 mOsM
initial osmolari.ty medium. A third sample was formed
by inoculating the standard osmolarity medium and,
after 88 hours of culture, -the 3.75 M salt solution
was added to a final concentration of ~00 mOsM. At
this time point, the cell density was determined indi-
~ ~rc~c~ rk
1 31 2030
--19--
cating that the culture contained ~ 1.2 x 106 vc/ml.The cells in each of the three cultures were cultured
for 9 days, during which time the cell viability and
cell density levels were monitored. The IgM titers
were determined for each of the three experimental
runs. The results of these experimental runs ~re pro-
vided in Figure 1 and in Table 1 below. As indicated
therein, a twofold increase in final IgM titers over
the control (~90 mg/L) was correlated with prolonged
viability and increased syecific IgM production rates
in 400 mOsM cultures where growth rate and cell den-
sity are reduced.
1 31 ~0~
--~o--
TABLE I
D-234 Summary Table
300 mOsM 400 mOsM "Add Salt~
Control Inikial (at 88 Hours)
Maximum Total 23 12 22
Cell Density
(10 /ml)
Maximum Viable 15 7.5 14
Cell Density
(105/ml)
Ave. Expo- 0.033 0.028 0.032
nential Growth (0-66 hr) (0-89 hr) (0-89 hr)
Rate mu (1/hr)
Final IgM 41 88 58
Concentration
(mg/L)
Ave. Exponen- 0.24 0.56 0.40
tial IgM Produc-
tion Rate
(mg/109/hr)
For the 400 mOsM/kg initial culture, expo-
nential growth rate "mu" and maximum cell density were
reduced, which was indicative of solute s-tress. The
duration of the culture was increased in the h~gh
osmolarity culture, and the specific IgM productivity
rate was twofold to thxeefold higher than the control.
The extra IgM over and above the control was produced
after the peak in viable cell density.
13120~)
-21-
A 1.5-fold increase in final IgM -titer to
~58 mg/L was observed in the culture where salt was
added at 88 hours. Specific IgM production rates
increased from one day after salt addition into the
viable cell decline (versus the control, where produc-
tion rate declined aftex the viable cell peak)l even
though there appeared to be little, if any, difference
in the growth curve compared to the control.
For the D-234 cell line, salt addition near
the peak viable cell density has an IgM production
enhancing effect in the decline phase without any
extension of the viable cell curve. This suggests
that specific IgM produc-tion rates can be increased
without slowing growth (and limiting ultimate cell
densities) early in culture. However, for D-234,
final titers are not as high as those achieved in slow
growing (limited cell density) cultures planted in
high osmolarity medium.
Example 3
Effect of Inoculation Density and
Timinq of Salt Addition
Using the methods described in the foregoing
examples, the effects of initial inoculation density
of D-234 on the specific cell productivity and timing
of the salt addition were explored.
A control was run at the standard osmolarity
of 300 mOsM medium using 5 x 104 planted cultures.
These cells exhibited good growth, bu-t viable cell
densities were lower than that produced for the 1 x
cultures (and total cell density of 1.6 versus 1.9
million) with an extension of the viable phase from
six to seven days. However, final IgM titers were
similar. At 370 mOsM, 5 x 10 cells/ml inoculated
cultures resulted in significant growth slowing and
-22- 1312~
lowerlng of viable cell density and titers, about half
compared with 1 x 105 planted cultures.
Various solute stress conditions were tested
using the 5 x 104 inoculation density cul-ture. Titers
for 300, 340 and 370 cultures were 40, 75, and 35 mg/
L, respectively. It was found that adding salt at day
one instead of at day zero to the 370 mOsM allowed the
5 x 104 culture to reach viable cell densities (5 x
105 cells/ml) and a titer (65 mg/L IgM) approaching
the 1 x 105, 370 culture values (6 x 105 cells/ml and
75 mg/L IgM).
From the results of the previous experimen-t,
3~0 and 370 mOsM were chosen as osmolarities to test
with salt added on day 0, 1, 2, or 3. The results
indicated that adding salt at different times to the
370 mOsM culture resulted in a slight increase (60 to
65 mg/L final IgM) in final titer concentration.
For the 340 mOsM culture, the addition of
salt at day 1 and day 2 led to higher titers (~110 mg/
20 L) than did day 3 addition (~90 mg/L) or day 0 (~70
mg/L).
Ex~ple 4
Effect of Salt Addition on T-88 Growth and
IqM Production
T-88 cells were grown in replicate 100 ml
working volume shake flasks of HL-1 media with 0.1~
w/v Pluronic~ polyol E'-68, 8 mM glutamine and 5~ added
fetal calf serum at 300 mOsM (control); 340 mOsM;
400 mOsM; and 450 mOsM. Like the above examples,
osmolality was increased by the addi-tion of a 3.75 M
sal-t solution with a 27:1 molar ratio NaCl:KCl. The
cultures were grown for 7 days, during which -time the
cell density and cel:L viability were periodically
monitored.
-23- l 3 1 ~0~()
Comple-te growth curves were generated for
the control and for the 400 mOsM flasks. The 400 mOsM
growth curve showed slow growth and reduced cell den-
sity, therefore indicating solute stress had occurred.
The duration of the culture was extended, during which
IgM production over and above the control was ob-
tained. The specific IgM production rate was higher
at 400 mOsM over most of the culture period. Table 2
shown below, illustrates that a 30~ reduction in total
cell density and a 20 to 25% increase in final IgM
titer for the 400 and 450 mOsM shake flasks was
achieved. IgM produced per million cells from day
three to day four was about -two times higher at 400
and 450 mOsM compared with the control and 340 mOsM
treatment. Exponential phase doubling time (Td) for
the 400 mOsM treated flasks was higher than for the
control (27 versus 20 hours).
Table 2
T-88 ~ 5~ FCS Summary Table
Control
300 340 400 450
mOsM mOsM mOsM mOsM
Maximum Total 23 24 17 17
Cell Density
(105/ml)
Final IgM 37 35 43 46
Concentra-
tion (mg/L)
IgM Produced 6 7 15 11
per Million
Cells From Day
3 to Day 4
(ug/105 cells/
day)
Ave. Exponen- 0.037 0.034
tial Growth (Td 20) (Td 27
Rate mu (1/hr)
-24- l 31~030
Example 5
Effect of Lactate on D-234 Grow~h and IgM Production
This example describes the effect of sodium
lactate on growth, viability, and IgM production of D-
234.
Approximately 1 x 10 cells/ml of D-234 were
grown in 250 ml shake flasks (agitated at 100 rpm) in
HL-1 medium containin~ 0.1~ Pluronic~ polyol F-68 and
B mM glutamine. A l M stock solution of sodium lac-
tate (pH 7.4) in HL-1 was added to the medium. A pre-
liminary screen of -the effect of a broad range of
sodium lactate concentrations (0-100 mM) on D-234
growth and IgM production was run. It was determined
that growth was greatly inhibited by levels of added
lactate above 40 mM. Cell densities at day four were
reduced at all levels of lactate tested with
critical drop between 40 and 60 mM.
The results of this experiment are given in
Table 3 below.
Table 3
Effect of Na Lactate on D-234 Growth
and IqM Production
Initial
Lactate Total Cell Density IgM
mM 1 x 105/ml (% Viability) uq/ml
Day 2 Day 4 Day 4 ~ 7
0 4.7 (95)21.0 (90) 10 24
5.6 (96)15.0 (92) 20 35
5.1 (92)12.9 (89) 22 54
~0 2.2 (93)4.1 (87) 19 61
2.1 (81)2~6 (65) 15 28
100 2.2 (72)2.0 (50) 11 1~
-25 13~2030
The results indicate that the production of
IgM by D-234 was increased with increasing concentr~-
tions of sodium lactate up to 60 mM where growth was
extremely inhibited, and IgM production peaked at 61
ug/ml compared to the control at 24 ug/ml. Even at 80
mM added lactate, the level of IgM produced was
similar to that seen for the control, even though the
cell density was only 12% of the control. Specific
(per cell) productivity was increased up to 14-fold
(at 60 mM added lactate).
Example 6
Effect of NH4Cl on D-234 Growth and IqM Production
The hybridoma D-234 was grown in HL-1 serum-
free medium supplemented with 0.1% Pluronic:~ polyol F-
6~, 10 mM glutamine and 10 mM NH4Cl. A control wasalso run without NH4Cl. One hundred ml cultures in
250 ml shake flasks were inoculated at an initial den-
sity of 1 x 105 viable cells/ml (91% viability).
As illustrated in Figure 2, the addition of
mM NH4Cl inhibited the growth, reduced both
viability and the maximum total cell density of the
culture (2.3 x 106/ml for the control vs 1.1 x 106/ml
when 10 mM NH4Cl was added). However, this stress
condition prolonged the stationary/decline phase and
resulted in a 2-fold increase in the production of
IgM.
Example 7
Effect of Hiqh Glucose Concentration
on Antibody Production
- 30 ~ The hybridoma D-234 was grown in HL-l medium
(Ventrex) which already contains 5.5 g/l. A 500 g/l
` stock solution of glucose was used -to increase the
glucose level of the HL-l medium. The -total glucose
~ `~rCyG/l~ rnci~k
26 ~ 1 31 2 030
levels tested in this example were 5.5 (control), 10.5,
1505, and 25.5 g/l.
The 10.5 g/l glucose culture grew more slowly than
the control and began to die sooner. While the control
reached a maximum of 8.7 x 105 viable cells/ml, the 10.5 g/l
stressed culture reached 7.1 x 105 viable cells/ml.
However, the death phase of this culture was longer than the
control resulting in higher antibody production: 85 versus
67 mg/l.
The 15.5 g/l glucose culture proved to be very
stressful for D-234 resultiny in a low maximum viable cell
density (4.3 x 105 viable cells/ml) and producing IgM at 50
mg/l. The 25.5 g/l glucose condition proved to be lethal.
Deposition~of Cultures
The hybridomas used in the above examples to
illustrate the method of the present invention were
deposited in and accepted by the American Type Culture
Collection (ATCC), 12301 Parklawn Drive, Rockville,
Maryland, USA, under the terms of the Budapest Treaty. In
addition, the mouse x human fusion partner F3B6 adapted to
99% serum-free medium which partner was the source of these
hybridomas was similarly deposited with the ATCC. The
deposit dates and the accession numhers are given below:
Culture Deposit Date Accession No.
D-234 10 August 1984 HB-8598
D-234 17 September 1987 HB-9543
T-88 19 May 1987 HB-9431
F3B6 18 April 1985 HB-8785
The deposits above were made pursuant to a contract
between the ATCC and the assignee of this
. ` ',;t
`: 'i
~...... ..
~ 31 2030
-27-
patent application, Cetus Corporation. The contract
with ATCC provides for permanent availability of the
progeny of these cell lines to the public on the issu-
ance of the U.S. patent describing and identifying the
deposit or the publications or upon the laying open to
the public of any U.S. or for~ign patent application,
whichever comes first, and for availability of the
progeny of these cell lines to one determined by th~
U.S. Commissioner of Paten-ts and Trademarks to be
entitled thereto according to 35 USC 122 and the
Commissioner's rules pursuant thereto (including 37
CFR 1.14 with particular reference to 886 OG 638).
The assignee of the present application has agreed
that if the cell lines on deposit should die or be
lost or destroyed when cultivated under suitable con-
ditions, they will be promptly replaced on notifica-
tion with a viable culture of the same cell line.
Samples o~ the 454A12 hybridomas had been
deposited with In Vitro International Inc., (formerly at
7885 JacXson Road, Suite 4, Ann Arbor, Michigan 48103,
U.S.A., currently at 611 P. Hammonds ~erry Road,
Linthicum, Maryland 21090, U.S.A., telephone number
~301)789-3636) on June 18, 1985, under the Accession No.
IVI10075. This deposit was made under the Budapest Treaty
and will be maintained and made accessible according to
the provisions thereof.
28 - l 3l 2 03 n
SUPP~EMENTARY DISCLOSURE
To the Principal Disclosure is added Fig. 3 of the drawings
and Examples 8 and 9.
In the drawings:
Figure 3 shows the effect of sodium chloride on
specific production rate of IgG antibody hybridoma ~54A12.
Exarnple 8
~ffect of PolyPropylene Glycol on IqG Production
The following experiment showed that when
polypropylene glycol (PPG) was added to hybridoma 454Al2
cell culture, it increased the IyG production of khe
hybridoma by 40%. Though PPG limited the maximum cell
density achievable by the culture, it slowed the decline in
culture viability a~ter the peak density had been attained.
The hybridoma 454A12 was grown in 125 ml shake flasks
filled to 50 ml with HL~l and 4mM glutamine. The test
sample contained 8~1/L of polypropylene glycol whereas the
control was without the PPG.
It was observed that the test sample exhibited an
exponential phase growth rate similar to the control at
0.054 hour l. However, the test sample experienced a lag in
growth of one day, and a higher exponential phase death rate
of 0.0059 hr~l. Additionally, the test sample had a lower
maximum cell density than the control. The test sample
reached a maximum cell density of only 1.3 million cells/ml,
whereas the control reached a maximum cell density of 2
million cells/ml.
Beyond the maximum cell density peak, the decline in
cell viability in the test sample was slower than in the
control. The test sample y:ielded a final IgG concentration
- 29 - l 3 1 203Q
of 63 ~g/ml, which was about 40~ higher than the control,
which ~ielded 46 ~g/ml of IgG.
Example 9
Effect of Sodium Chloride on I~G Production
In another experiment, the 454A12 hybridomas were
grown in shake flasks with commercially available HL~1
medium (Ventrex) supplemented with 8mM glutamine.
Osmolality of the standard (control) HL-l medium was 300
mOsmol/kg. In the test sample, the osmolality was increased
to 400 mOsmol/kg using sodium chloride. The result of
solute stress in the test sample was evidenced by a 50
decrease in maximum cell density. In the stressed
condition, the specific IgG production rate per cell
increased throughout the culture period. ~he increase in
specific productivity was greatest during the post
exponential phase period of the culture when specific
productivity was more than 60~ higher under the stressed
condition (Figure 3).
