U.S. patent application number 13/610370 was filed with the patent office on 2013-03-14 for dry powder cells and cell culture reagents and methods of production thereof.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. The applicant listed for this patent is William Biddle, Richard FIKE, William Whitford. Invention is credited to William Biddle, Richard FIKE, William Whitford.
Application Number | 20130065300 13/610370 |
Document ID | / |
Family ID | 27365699 |
Filed Date | 2013-03-14 |
United States Patent
Application |
20130065300 |
Kind Code |
A1 |
FIKE; Richard ; et
al. |
March 14, 2013 |
DRY POWDER CELLS AND CELL CULTURE REAGENTS AND METHODS OF
PRODUCTION THEREOF
Abstract
The present invention relates generally to nutritive medium,
medium supplement, media subgroup and buffer formulations.
Specifically, powdered nutritive medium, supplement, subgroup
formulations, cell culture media comprising all of the necessary
nutritive factors for in vitro cell cultivation, buffer
formulations that produce particular ionic and pH conditions upon
reconstitution with a solvent are provided. Particularly, methods
of production of these media, supplement, subgroup, buffer
formulations and kits, and methods for the cultivation of
prokaryotic and eukaryotic cells using these dry powdered nutritive
media, supplement, subgroup and buffer formulations are provided.
Methods of producing sterile, powdered media or supplement (e.g.,
powdered FBS, powdered transferrin, powdered insulin, powdered
organ extracts, powdered growth factors), media subgroup and buffer
formulations by gamma irradiation are provided. Methods for
producing dry cell powders, comprising spray-drying a cell
suspension, and cells, media, media supplement, media subgroup and
buffer powders produced by these methods are provided.
Inventors: |
FIKE; Richard; (Clarence,
NY) ; Whitford; William; (Logan, UT) ; Biddle;
William; (Buffalo, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FIKE; Richard
Whitford; William
Biddle; William |
Clarence
Logan
Buffalo |
NY
UT
NY |
US
US
US |
|
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
27365699 |
Appl. No.: |
13/610370 |
Filed: |
September 11, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13452498 |
Apr 20, 2012 |
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13610370 |
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09606314 |
Jun 29, 2000 |
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13452498 |
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09023790 |
Feb 13, 1998 |
6383810 |
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09606314 |
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60040314 |
Feb 14, 1997 |
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60058716 |
Sep 12, 1997 |
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60062192 |
Oct 16, 1997 |
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Current U.S.
Class: |
435/346 ; 422/22;
435/255.7; 435/352; 435/353; 435/354; 435/358; 435/364; 435/365;
435/366; 435/367; 435/369; 435/404; 435/431 |
Current CPC
Class: |
C12N 5/0018 20130101;
C12N 5/0025 20130101; C12N 1/20 20130101; C12N 1/14 20130101; C12N
1/16 20130101 |
Class at
Publication: |
435/346 ;
435/255.7; 435/404; 435/431; 435/358; 435/369; 435/364; 435/365;
435/352; 435/366; 435/354; 435/353; 435/367; 422/22 |
International
Class: |
C12N 5/07 20100101
C12N005/07; A61L 2/08 20060101 A61L002/08; C12N 5/12 20060101
C12N005/12; C12N 1/16 20060101 C12N001/16; C12N 5/04 20060101
C12N005/04 |
Claims
1-9. (canceled)
92. An agglomerated eukaryotic cell culture medium, wherein the
medium is a complete media, a media subgroup, a supplement, or a
buffer and wherein said medium has been sterilized using gamma
irradiation.
93. The medium of claim 92, wherein the medium has been sterilized
after packaging.
94. The medium of claim 92 that is capable of culturing a
eukaryotic cell after reconstitution with a solvent.
95. The medium of claim 94, and a eukaryotic cell.
96. The medium of claim 95 wherein said cell is either a plant,
animal or a yeast cell.
97. The medium of claim 96, wherein the animal cell is selected
from the group consisting of CHO, 293, VERO, BHK, SP2/0, MDBK, COS,
AE-1, L5.1, PER-C6, hybridoma cells and HeLa cells.
98. The medium of claim 92 wherein the gamma irradiation is done at
least 25 kGy for several days.
99. The medium of claim 94 wherein the solvent is water.
100. The medium of claim 94 wherein the solvent is an aqueous
buffer.
101. The medium of claim 98 wherein the gamma irradiation
inactivates bacteria, fungi, spores and viruses present in the
medium, supplement or buffer.
102. A method of making the medium of claim 92, comprising
subjecting an agglomerated eukaryotic medium to gamma
irradiation.
103. The method of claim 102 wherein the inactivation renders the
bacteria, fungi, spores and virus incapable of replication.
104. The method of claim 103 wherein the gamma-irradiation is done
at a total dosage of about 10-100 kGy.
105. The method of claim 104 wherein the total dosage is about
15-75 kGy.
106. The method of claim 104 wherein the total dosage is about
20-40 kGy.
107. The method of claim 104 wherein the gamma-irradiation is done
for about 1 hour to about 7 days, or for about 1 hour to about 5
days, or for about 1 hour to about 3 days, or for about 1 hour to
about 24 hours.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Nos. 60/040,314, filed Feb. 14, 1997, 60/058,716, filed
Sep. 12, 1997, and 60/062,192, filed Oct. 16, 1997, the disclosures
of which are entirely incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to cells, nutritive
media, media supplements, media subgroups and buffer formulations.
Specifically, the present invention provides dry powder nutritive
medium formulations, particularly cell culture medium formulations,
comprising all of the necessary nutritive factors that facilitate
the in vitro cultivation of cells, and methods of production of
these media formulations. The invention also relates to methods of
producing dry powder media supplements, such as dry powder sera
(e.g., fetal bovine serum). The invention also relates to dry
powder buffer formulations that produce particular ionic and pH
conditions upon rehydration. The invention also relates to methods
of producing dry powder cells, such as prokaryotic (e.g.,
bacterial) and eukaryotic (e.g., fungal (especially yeast), animal
(especially mammalian) and plant cells). The invention also relates
to methods of preparing sterile dry powder nutritive media, media
supplements (particularly dry powder sera), media subgroups and
buffer formulations. The invention also relates to dry powder
nutritive media, media supplements, media subgroups, buffer
formulations and cells prepared by these methods. The present
invention also relates to kits and methods for cultivation of
prokaryotic and eukaryotic cells using these dry powder nutritive
media, media supplements, media subgroups and buffer
formulations.
BACKGROUND OF THE INVENTION
Cell Culture Media
[0003] Cell culture media provide the nutrients necessary to
maintain and grow cells in a controlled, artificial and in vitro
environment. Characteristics and compositions of the cell culture
media vary depending on the particular cellular requirements.
Important parameters include osmolality, pH, and nutrient
formulations.
[0004] Media formulations have been used to cultivate a number of
cell types including animal, plant and bacterial cells. Cells
cultivated in culture media catabolize available nutrients and
produce useful biological substances such as monoclonal antibodies,
hormones, growth factors, viruses and the like. Such products have
therapeutic applications and, with the advent of recombinant DNA
technology, cells can be engineered to produce large quantities of
these products. Thus, the ability to cultivate cells in vitro is
not only important for the study of cell physiology, but is also
necessary for the production of useful substances which may not
otherwise be obtained by cost-effective means.
[0005] Cell culture media formulations have been well documented in
the literature and a number of media are commercially available. In
early cell culture work, media formulations were based upon the
chemical composition and physicochemical properties (e.g.,
osmolality, pH, etc.) of blood and were referred to as
"physiological solutions" (Ringer, S., J. Physiol. 3:380-393
(1880); Waymouth, C., In: Cells and Tissues in Culture, Vol. 1,
Academic Press, London, pp. 99-142 (1965); Waymouth, C., In Vitro
6:109-127 (1970)). However, cells in different tissues of the
mammalian body are exposed to different microenvironments with
respect to oxygen/carbon dioxide partial pressure and
concentrations of nutrients, vitamins, and trace elements;
accordingly, successful in vitro culture of different cell types
will often require the use of different media formulations. Typical
components of cell culture media include amino acids, organic and
inorganic salts, vitamins, trace metals, sugars, lipids and nucleic
acids, the types and amounts of which may vary depending upon the
particular requirements of a given cell or tissue type. Often,
particularly in complex media compositions, stability problems
result in toxic products and/or lower effective concentrations of
required nutrients, thereby limiting the functional life-span of
the culture media. For instance, glutamine is a constituent of
almost all media that are used in culturing of mammalian cells in
vitro. Glutamine decomposes spontaneously into pyrolidone
carboxylic acid and ammonia. The rate of degradation can be
influenced by pH and ionic conditions but in cell culture media,
formation of these breakdown products often cannot be avoided
(Tritsch et al, Exp. Cell Res. 28:360-364 (1962)).
[0006] Wang et al. (In Vitro 14(8):715-722 (1978)) have shown that
photoproducts such as hydrogen peroxide, which are lethal to cells,
are produced in Dulbecco's Modified Eagle's Medium (DMEM).
Riboflavin and tryptophan or tyrosine are components necessary for
formation of hydrogen peroxide during light exposure. Since most
mammalian culture media contain riboflavin, tyrosine and
tryptophan, toxic photoproducts are likely produced in most cell
culture media.
[0007] To avoid these problems, researchers make media on an "as
needed" basis, and avoid long term storage of the culture media.
Commercially available media, typically in dry power form, serves
as a convenient alternative to making the media from scratch, i.e.,
adding each nutrient individually, and also avoids some of the
stability problems associated with liquid media. However, only a
limited number of commercial culture media are available, except
for those custom formulations supplied by the manufacturer.
[0008] Although dry powder media formulations may increase the
shelf-life of some media, there are a number of problems associated
with dry powdered media, especially in large scale application.
Production of large media volumes requires storage facilities for
the dry powder media, not to mention the specialized media kitchens
necessary to mix and weigh the nutrient components. Due to the
corrosive nature of dry powder media, mixing tanks must be
periodically replaced.
[0009] Typically, cell culture media formulations are supplemented
with a range of additives, including undefined components such as
fetal bovine serum (FBS) (10-20% v/v) or extracts from animal
embryos, organs or glands (0.5-10% v/v). While FBS is the most
commonly applied supplement in animal cell culture media, other
serum sources are also routinely used, including newborn calf,
horse and human. Organs or glands that have been used to prepare
extracts for the supplementation of culture media include
submaxillary gland (Cohen, S., J. Biol. Chem. 237:1555-1565
(1961)), pituitary (Peehl, D. M., and Ham, R. G., In Vitro
16:516-525 (1980); U.S. Pat. No. 4,673,649), hypothalamus (Maciag,
T., et al., Proc. Natl. Acad. Sci. USA 76:5674-5678 (1979);
Gilchrest, B. A., et al, J. Cell. Physiol. 120:377-383 (1984)),
ocular retina (Barretault, D., et al., Differentiation 18:29-42
(1981)) and brain (Maciag, T., et al., Science 211:1452-1454
(1981)). These types of chemically undefined supplements serve
several useful functions in cell culture media (Lambert, K. J. et
al., In: Animal Cell Biotechnology, Vol. 1, Spier, R. E. et al.,
Eds., Academic Press New York, pp. 85-122 (1985)). For example,
these supplements provide carriers or chelators for labile or
water-insoluble nutrients; bind and neutralize toxic moieties;
provide hormones and growth factors, protease inhibitors and
essential, often unidentified or undefined low molecular weight
nutrients; and protect cells from physical stress and damage. Thus,
serum or organ/gland extracts are commonly used as relatively
low-cost supplements to provide an optimal culture medium for the
cultivation of animal cells.
Methods of Production of Culture Media
[0010] Culture media are typically produced in liquid form or in
powdered form. Each of these forms has particular advantages and
disadvantages.
[0011] For example, liquid culture medium has the advantage that it
is provided ready-to-use (unless supplementation with nutrients or
other components is necessary), and that the formulations have been
optimized for particular cell types. Liquid media have the
disadvantages, however, that they often do require the addition of
supplements (e.g., L-glutamine, serum, extracts, cytokines, lipids,
etc.) for optimal performance in cell cultivation. Furthermore,
liquid medium is often difficult to sterilize economically, since
many of the components are heat labile (thus obviating the use of
autoclaving, for example) and bulk liquids are not particularly
amenable to penetrating sterilization methods such as gamma or
ultraviolet irradiation; thus, liquid culture media are most often
sterilized by filtration, which can become a time-consuming and
expensive process. Furthermore, production and storage of large
batch sizes (e.g., 1000 liters or more) of liquid culture media are
impractical, and the components of liquid culture media often have
relatively short shelf lives.
[0012] To overcome some of these disadvantages, liquid culture
medium can be formulated in concentrated form; these media
components may then be diluted to working concentrations prior to
use. This approach provides the capability of making larger and
variable batch sizes than with standard culture media, and the
concentrated media formulations or components thereof often have
longer shelf-life (see U.S. Pat. No. 5,474,931, which is directed
to culture media concentrate technology). Despite these advantages,
however, concentrated liquid media still have the disadvantages of
their need for the addition of supplements (e.g., FBS, L-glutamine
or organ/gland extracts), and may be difficult to sterilize
economically.
[0013] As an alternative to liquid media, powdered culture media
are often used. Powdered media are typically produced by admixing
the dried components of the culture medium via a mixing process,
e.g., ball-milling, or by lyophilizing pre-made liquid culture
medium. This approach has the advantages that even larger batch
sizes may be produced, the powdered media typically have longer
shelf lives than liquid media, and the media can be sterilized by
irradiation (e.g., gamma or ultraviolet irradiation) or ethylene
oxide permeation after formulation. However, powdered media have
several distinct disadvantages. For example, some of the components
of powdered media become insoluble or aggregate upon lyophilization
such that resolubilization is difficult or impossible. Furthermore,
powdered media typically comprise fine dust particles which can
make them particularly difficult to reconstitute without some loss
of material, and which may further make them impractical for use in
many biotechnology production facilities operating under GMP/GLP,
USP or ISO 9000 settings. Additionally, many of the supplements
used in culture media, e.g., L-glutamine and FBS, cannot be added
to the culture medium prior to lyophilization or ball-milling due
to their instability or propensity to aggregate upon concentration
or due to their sensitivity to shearing by processes such as
ball-milling. Finally, many of these supplements, particularly
serum supplements such as FBS, show a substantial loss of activity
or are rendered completely inactive if attempts are made to produce
powdered supplements by processes such as lyophilization.
[0014] Thus, there exists a current need for rapidly dissolving
nutritionally complex stable dry powder nutritive media, media
supplements, media subgroups and buffers, which can be prepared in
variable bulk quantities and which are amenable to sterilization
particularly by ionizing or ultraviolet irradiation.
SUMMARY OF THE INVENTION
[0015] The present invention provides methods for the production of
nutritive media, media supplement, media subgroup and buffer
powders comprising agglomerating a dry powder nutritive media,
media supplement, media subgroup or buffer with a solvent. The
invention also relates to methods for the production of powdered
nutritive media, media supplements, media subgroups, and buffers,
comprising spray-drying a liquid nutritive medium, medium
supplement, medium subgroup or buffer under conditions sufficient
to produce their dry powder counterparts. Such conditions may, for
example, comprise controlling heat and humidity until the powdered
media, media supplement, media subgroup or buffer is formed.
According to the invention, the method may further comprise
sterilizing the nutritive media, media supplement, media subgroup
or buffer powder, which may be accomplished prior to or after
packaging the powder. In particularly preferred methods, the
sterilization is accomplished after packaging of the powder by
irradiation of the packaged powder with gamma rays.
[0016] Particularly preferred nutritive medium powders that may be
produced according to the invention include culture medium powders
selected from the group consisting of a bacterial culture medium
powder, a yeast culture medium powder, a plant culture medium
powder and an animal culture medium powder.
[0017] Particularly preferred media supplements that may be
produced by the methods of the invention include: powdered animal
sera, such as bovine sera (e.g., fetal bovine, newborn calf or
normal calf sera), human sera, equine sera, porcine sera, monkey
sera, ape sera, rat sera, murine sera, rabbit sera, ovine sera and
the like; cytokines (including growth factors (such as EGF, aFGF,
bFGF, HGF, IGF-1, IGF-2, NGF and the like), interleukins,
colony-stimulating factors and interferons); attachment factors or
extracellular matrix components (such as collagens, laminins,
proteoglycans, glycosaminoglycans, fibronectin, vitronectin and the
like); lipids (such as phospholipids, cholesterol, bovine
cholesterol concentrate, fatty acids, sphingolipids and the like);
and extracts of animal tissues, organs or glands (such as bovine
pituitary extract, bovine brain extract, chick embryo extract,
bovine embryo extract, chicken meat extract, achilles tendon and
extracts thereof) and the like). Other media supplements that may
be produced by the present methods include a variety of proteins
(such as serum albumins, particularly bovine or human serum
albumins; immunoglobulins and fragments or complexes thereof,
aprotinin; hemoglobin; haemin or haematin; enzymes (such as
trypsin, collagenases, pancreatinin or dispase); lipoproteins;
ferritin; etc.) which may be natural or recombinant; vitamins;
amino acids and variants thereof (including, but not limited to,
L-glutamine and cystine), enzyme co-factors and other components
useful in cultivating cells in vitro that will be familiar to one
of ordinary skill.
[0018] The nutritive media and media supplements prepared by the
invention may also comprise subgroups such as serum (preferably
those described above), L-glutamine, insulin, transferrin, one or
more lipids (preferably one or more phospholipids, sphingolipids,
fatty acids or cholesterol), one or more cytokines (preferably
those described above), one or more neurotransmitters, one or more
extracts of animal tissues, organs or glands (preferably those
described above), one or more proteins (preferably those described
above) or one or more buffers (preferably sodium bicarbonate), or
any combination thereof.
[0019] Buffer powders particularly suitable for preparation
according to the methods of the invention include buffered saline
powders; most particularly phosphate-buffered saline powders or
Tris-buffered saline powders.
[0020] The invention also provides nutritive medium powders, medium
supplement powders (including powders of the above-described
supplements) and buffer powders prepared according to these
methods.
[0021] The invention also relates to methods of preparing dried
cells, including prokaryotic (e.g., bacterial) and eukaryotic
(e.g., fungal (especially yeast), animal (especially mammalian,
including human) and plant) cells, comprising obtaining a cell to
be dried, contacting the cell with one or more stabilizers (e.g.; a
polysaccharide such as trehalose), forming an aqueous suspension
comprising the cell, and spray-drying the cell suspension under
conditions favoring the production of a dried powder. The invention
also relates to dried cell powders produced by these methods.
[0022] The invention further relates to methods of preparing
sterile powdered culture media, media supplements, media subgroups
and buffers. One such method comprises exposing the above-described
powdered culture media, media supplements, media subgroups and
buffers to .gamma. irradiation such that bacteria, fungi, spores
and viruses that may be resident in the powders are rendered
incapable of replication. In a preferred such method, the powdered
media, media supplements, media subgroups and buffers are .gamma.
irradiated at a total dosage of about 10-100 kilograys (kGy),
preferably a total dosage of about 15-75 kGy, 15-50 kGy, 15-40 kGy
or 20-40 kGy, more preferably a total dosage of about 20-30 kGy,
and most preferably a total dosage of about 25 kGy, for about 1
hour to about 7 days, preferably for about 1 hour to about 5 days,
more preferably for about 1 hour to about 3 days, about 1 hour to
about 24 hours or about 1-5 hours, and most preferably about 1-3
hours. The invention also relates to sterile powdered culture
media, media supplements, media subgroups and buffers produced by
these methods.
[0023] The invention further provides methods of culturing a cell
comprising reconstituting the nutritive media, media supplement,
media subgroup or buffer of the invention with a solvent, which
preferably comprises serum or water, and contacting the cell with
the reconstituted nutritive media, media supplement, media subgroup
or buffer under conditions favoring the cultivation of the cell.
Any cell may be cultured according to the present methods,
particularly bacterial cells, yeast cells, plant cells or animal
cells. Preferable animal cells for culturing by the present methods
include insect cells (most preferably Drosophila cells, Spodoptera
cells and Trichoplusa cells), nematode cells (most preferably C.
elegans cells) and mammalian cells (most preferably CHO cells, COS
cells, VERO cells, BHK cells, AE-1 cells, SP2/0 cells, L5.1 cells,
hybridoma cells or human cells). Cells cultured according to this
aspect of the invention may be normal cells, diseased cells,
transformed cells, mutant cells, somatic cells, germ cells, stem
cells, precursor cells or embryonic cells, any of which may be
established cell lines or obtained from natural sources.
[0024] The invention is further directed to kits for use in the
cultivation of a cell. Kits according to the invention may comprise
one or more containers containing one or more of the nutritive
media powders, media supplement powders, media subgroup powders or
buffer powders of the invention, or any combination thereof. The
kits may also comprise one or more cells or cell types, including
the dried cell powders of the invention.
[0025] Other preferred embodiments of the present invention will be
apparent to one of ordinary skill in light of the following
drawings and description of the invention, and of the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 is a histogram of a densitometric scan of SDS-PAGE of
samples of fetal bovine serum (FBS) prepared in powdered form by
the methods of the invention (FIG. 1A) and conventional liquid FBS
(FIG. 1B).
[0027] FIG. 2 is a composite of line graphs of growth (FIG. 2A) and
passage success (FIG. 2B) of SP2/0 cells in Dulbecco's Modified
Eagle's Medium (DMEM) supplemented with 2% (w/v) FBS prepared in
powdered form by the agglomeration methods of the invention.
[0028] FIG. 3 is composite of histograms of spectrophotometric
scans (A=200-350 nm) of powdered fetal bovine serum (FBS) prepared
by spray-drying according to the methods of the invention (FIG. 3A)
or of standard liquid FBS (FIG. 3B).
[0029] FIG. 4 is a composite of line graphs showing the pH
titration (buffer capacity), on two different dates (FIGS. 4A and
4B), of various dry powdered media (DPM) prepared by the methods of
the invention or by ball-milling, with or without the addition of
sodium bicarbonate.
[0030] FIG. 5 is a composite of bar graphs showing the effect of
agglomeration on dissolution rates (in water) of Opti-MEM I.TM.
(FIG. 5A) or DMEM (FIG. 5B). Media were agglomerated with water or
FBS as indicated.
[0031] FIG. 6 is a composite of line graphs showing growth over
seven days of SP2/0 cells in agglomerated Opti-MEM I.TM. (FIG. 6A)
or DMEM (FIG. 6B), both containing 2% FBS.
[0032] FIG. 7 is a composite of line graphs showing growth over
seven days of SP2/0 cells (FIG. 7A), AE-1 cells (FIG. 7B) and L5.1
cells (FIG. 7C) in agglomerated DMEM containing 10% FBS.
[0033] FIG. 8 is a composite of line graphs showing passage success
of SP2/0 cells in Opti-MEM I.TM. (FIG. 8A) or DMEM (FIG. 8B),
agglomerated with either water or FBS, supplemented with 2%
FBS.
[0034] FIG. 9 is a composite of line graphs showing passage success
of SP2/0 cells (FIG. 9A), AE-1 cells (FIG. 9B) and L5.1 cells (FIG.
9C) in DMEM agglomerated with FBS and sodium bicarbonate and
supplemented with 10% FBS.
[0035] FIG. 10 is a line graph showing the growth of SP2/0 cells
over four passages in standard water-reconstituted powdered culture
media (control media), or in agglomerated powdered culture media
prepared in large-scale amounts according to the methods of the
invention. Results are shown for control media (.quadrature.),
water-agglomerated powdered culture media of the invention
(.diamond-solid.) and water-agglomerated auto-pH powdered culture
media (containing sodium bicarbonate) of the invention
(.box-solid.).
[0036] FIG. 11 is a line graph of AE-1 cells cultured over six or
seven days in medium containing 2% (.tangle-solidup.) or 10%
(.diamond-solid.) liquid fetal bovine serum (FBS), or 2% () or 10%
(.box-solid.) powdered FBS prepared by the spray-drying methods of
the invention. Duplicate experiments are shown in FIGS. 11A and
11B.
[0037] FIG. 12 is a line graph of SP2/0 cells cultured over seven
days in medium containing 2% (.tangle-solidup.) or 10%
(.diamond-solid.) liquid FBS, or 2% () or 10% (.box-solid.)
powdered FBS prepared by the spray-drying methods of the invention.
Duplicate experiments are shown in FIGS. 12A and 12B.
[0038] FIG. 13 is a line graph of AE-1 cell growth over four
passages in media containing 5% liquid FBS (.diamond-solid.) or 5%
powdered FBS prepared by the spray-drying methods of the invention
(.box-solid.).
[0039] FIG. 14 is a line graph indicating the effect of .gamma.
irradiation and agglomeration on the growth of SP2/0 cells over
five days.
[0040] FIG. 15 is a bar graph indicating the effect of .gamma.
irradiation on the growth of VERO cells in agglomerated culture
media.
[0041] FIG. 16 is a series of line graphs indicating the effect of
.gamma. irradiation on the ability of transferrin to support the
growth of 293 cells over four passages. In each graph, cells were
cultured in standard serum-free 293 medium (.diamond-solid.), in
medium without transferrin (.box-solid.), in medium containing
powdered transferrin that had been .gamma. irradiated at
-70.degree. C. (.tangle-solidup.) or room temperature (), or in
medium containing powdered transferrin that had not been .gamma.
irradiated but that had been stored at -70.degree. C. () or at room
temperature ( ). Results for each data point are the averages of
duplicate flasks.
[0042] FIG. 16A: passage 1 cells;
[0043] FIG. 16B: passage 2 cells;
[0044] FIG. 16C: passage 3 cells;
[0045] FIG. 16D: passage 4 cells.
[0046] FIG. 17 is a series of bar graphs indicating the effect of
.gamma. irradiation, under different irradiation conditions, on the
ability of FBS to support growth of anchorage-independent cells
(FIGS. 17A and 17B) and anchorage-dependent cells (FIGS. 17C and
17D) at first (Px1), second (Px2) and third (Px3) passages.
[0047] FIG. 17A: SP2/0 cells;
[0048] FIG. 17B: AE-1 cells;
[0049] FIG. 17C: VERO cells;
[0050] FIG. 17D: BHK cells.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0051] In the description that follows, a number of terms
conventionally used in the field of cell culture media are utilized
extensively. In order to provide a clear and consistent
understanding of the specification and claims, and the scope to be
given such terms, the following definitions are provided.
[0052] The term "powder" as used herein refers to a composition
that is present in granular form, which may or may not be complexed
or agglomerated with a solvent such as water or serum. The term
"dry powder" may be used interchangeably with the term "powder,"
however, "thy powder" as used herein simply refers to the gross
appearance of the granulated material and is not intended to mean
that the material is completely free of complexed or agglomerated
solvent unless otherwise indicated.
[0053] The term "ingredient" refers to any compound, whether of
chemical or biological origin, that can be used in cell culture
media to maintain or promote the growth of proliferation of cells.
The terms "component," "nutrient" and ingredient" can be used
interchangeably and are all meant to refer to such compounds.
Typical ingredients that are used in cell culture media include
amino acids, salts, metals, sugars, lipids, nucleic acids,
hormones, vitamins, fatty acids, proteins and the like. Other
ingredients that promote or maintain cultivation of cells ex vivo
can be selected by those of skill in the art, in accordance with
the particular need.
[0054] The term "cytokine" refers to a compound that induces a
physiological response in a cell, such as growth, differentiation,
senescence, apoptosis, cytotoxicity or antibody secretion. Included
in this definition of "cytokine" are growth factors, interleukins,
colony-stimulating factors, interferons and lymphokines.
[0055] By "cell culture" or "culture" is meant the maintenance of
cells in an artificial, e.g., an in vitro environment. It is to be
understood, however, that the term "cell culture" is a generic term
and may be used to encompass the cultivation not only of individual
prokaryotic (e.g., bacterial) or eukaryotic (e.g., animal, plant
and fungal) cells, but also of tissues, organs, organ systems or
whole organisms, for which the terms "tissue culture," "organ
culture," "organ system culture" or "organotypic culture" may
occasionally be used interchangeably with the term "cell
culture."
[0056] By "cultivation" is meant the maintenance of cells in an
artificial environment under conditions favoring growth,
differentiation or continued viability, in an active or quiescent
state, of the cells. Thus, "cultivation" may be used
interchangeably with "cell culture" or any of its synonyms
described above.
[0057] By "culture vessel" is meant a glass, plastic, or metal
container that can provide an aseptic environment for culturing
cells.
[0058] The phrases "cell culture medium," "culture medium" (plural
"media" in each case) and "medium formulation" refer to a nutritive
solution that supports the cultivation and/or growth of cells;
these phrases may be used interchangeably.
[0059] By "extract" is meant a composition comprising a
concentrated preparation of the subgroups of a substance, typically
formed by treatment of the substance either mechanically (e.g., by
pressure treatment) or chemically (e.g., by distillation,
precipitation, enzymatic action or high salt treatment).
[0060] By "enzymatic digest" is meant a composition comprising a
specialized type of extract, namely one prepared by treating the
substance to be extracted (e.g., plant components or yeast cells)
with at least one enzyme capable of breaking down the components of
the substance into simpler forms (e.g., into a preparation
comprising mono- or disaccharides and/or mono-, di- or
tripeptides). In this context, and for the purposes of the present
invention, the term "hydrolysate" may be used interchangeably with
the term "enzymatic digest."
[0061] The term "contacting" refers to the placing of cells to be
cultivated into a culture vessel with the medium in which the cells
are to be cultivated. The term "contacting" encompasses mixing
cells with medium, pipetting medium onto cells in a culture vessel,
and submerging cells in culture medium.
[0062] The term "combining" refers to the mixing or admixing of
ingredients in a cell culture medium formulation.
[0063] A cell culture medium is composed of a number of ingredients
and these ingredients vary from one culture medium to another. A
"1.times. formulation" is meant to refer to any aqueous solution
that contains some or all ingredients found in a cell culture
medium at working concentrations. The "1.times. formulation" can
refer to, for example, the cell culture medium or to any subgroup
of ingredients for that medium. The concentration of an ingredient
in a 1.times. solution is about the same as the concentration of
that ingredient found in a cell culture formulation used for
maintaining or cultivating cells in vitro. A cell culture medium
used for the in vitro cultivation of cells is a 1.times.
formulation by definition. When a number of ingredients are
present, each ingredient in a 1.times. formulation has a
concentration about equal to the concentration of those ingredients
in a cell culture medium. For example, RPMI-1640 culture medium
contains, among other ingredients, 0.2 g/L L-arginine, 0.05 g/L
L-asparagine, and 0.02 g/L L-aspartic acid. A "1.times.
formulation" of these amino acids contains about the same
concentrations of these ingredients in solution. Thus, when
referring to a "1.times. formulation," it is intended that each
ingredient in solution has the same or about the same concentration
as that found in the cell culture medium being described. The
concentrations of ingredients in a 1.times. formulation of cell
culture medium are well known to those of ordinary skill in the
art. See Methods For Preparation of Media, Supplements and
Substrate For Serum-Free Animal Cell Culture Allen R. Liss, N.Y.
(1984), which is incorporated by reference herein in its entirety.
The osmolality and/or pH, however, may differ in a 1.times.
formulation compared to the culture medium, particularly when fewer
ingredients are contained in the 1.times. formulation.
[0064] A "10.times. formulation" is meant to refer to a solution
wherein each ingredient in that solution is about 10 times more
concentrated than the same ingredient in the cell culture medium.
For example, a 10.times. formulation of RPMI-1640 culture medium
may contain, among other ingredients, 2.0 g/L L-arginine, 0.5 g/L
L-asparagine, and 0.2 g/L L-aspartic acid (compare 1.times.
formulation, above). A "10.times. formulation" may contain a number
of additional ingredients at a concentration about 10 times that
found in the 1.times. culture medium. As will be readily apparent,
"20.times. formulation," "25.times. formulation," "50.times.
formulation" and "100.times. formulation" designate solutions that
contain ingredients at about 20-, 25-, 50- or 100-fold
concentrations, respectively, as compared to a 1.times. cell
culture medium. Again, the osmolality and pH of the media
formulation and concentrated solution may vary. See U.S. Pat. No.
5,474,931, which is directed to culture media concentrate
technology.
Overview
[0065] The present invention is directed to methods of producing
nutritive media, media supplements, media subgroups or buffers.
Nutritive media, media supplements and media subgroups produced by
the present methods are any media, media supplement or media
subgroup (serum-free or serum-containing) which may be used to
support the growth of a cell, which may be a bacterial cell, a
fungal cell (particularly a yeast cell), a plant cell or an animal
cell (particularly an insect cell, a nematode cell or a mammalian
cell, most preferably a human cell), any of which may be a somatic
cell, a germ cell, a normal cell, a diseased cell, a transformed
cell, a mutant cell, a stem cell, a precursor cell or an embryonic
cell. Preferred such nutritive media include, but are not limited
to, cell culture media, most preferably a bacterial cell culture
medium, plant cell culture medium or animal cell culture medium.
Preferred media supplements include, but are not limited to,
undefined supplements such as extracts of bacterial, animal or
plant cells, glands, tissues or organs (particularly bovine
pituitary extract, bovine brain extract and chick embryo extract);
and biological fluids (particularly animal sera, and most
preferably bovine serum (particularly fetal bovine, newborn calf or
normal calf serum), horse serum, porcine serum, rat serum, murine
serum, rabbit serum, monkey serum, ape serum or human serum, any of
which may be fetal serum) and extracts thereof (more preferably
serum albumin and most preferably bovine serum albumin or human
serum albumin). Medium supplements may also include defined
replacements such as LipoMAX.RTM., OptiMAb.RTM., Knock-Out.TM. SR
(each available from Life Technologies, Inc., Rockville, Md.), and
the like, which can be used as substitutes for the undefined media
supplements described above. Such supplements may also comprise
defined components, including but not limited to, hormones,
cytokines, neurotransmitters, lipids, attachment factors, proteins
and the like.
[0066] Nutritive media can also be divided into various subgroups
(see U.S. Pat. No. 5,474,931) which can be prepared by, and used in
accordance with, the methods of the invention. Such subgroups can
be combined to produce the nutritive media of the present
invention.
[0067] By the methods of the present invention, any nutritive
media, media supplement, media subgroup or buffer may be produced
and stored for an extended period of time without significant loss
of biological and biochemical activity. By "without significant
loss of biological and biochemical activity" is meant a decrease of
less than about 30%, preferably less than about 25%, more
preferably less than about 20%, still more preferably less than
about 15%, and most preferably less than about 10%, of the
biological or biochemical activity of the nutritive media, media
supplement, media subgroup or buffer when compared to a freshly
made nutritive media, media supplement, media subgroup or buffer of
the same formulation. By an "extended period of time" is meant a
period of time longer than that for which a nutritive medium,
supplement, subgroup or buffer is stored when prepared by
traditional methods such as ball-milling. As used herein, an
"extended period of time" therefore means about 1-36 months, about
2-30 months, about 3-24 months, about 6-24 months, about 9-18
months, or about 4-12 months, under a given storage condition,
which may include storage at temperatures of about -70.degree. C.
to about 25.degree. C., about, 20.degree. C. to about 25.degree.
C., about 0.degree. C. to about 25.degree. C., about 4.degree. C.
to about 25.degree. C., about 10.degree. C. to about 25.degree. C.,
or about 20.degree. C. to about 25.degree. C. Assays for
determining the biological or biochemical activity of a nutritive
media, media supplement, media subgroup or buffer are well-known in
the art and are familiar to one of ordinary skill.
Formulation of Media, Media Supplements, Media Subgroups and
Buffers
[0068] Any nutritive media, media supplement, media subgroup or
buffer may be prepared by the methods of the present invention.
Particularly preferred nutritive media, media supplements and media
subgroups that may be prepared according to the invention include
cell culture media, media supplements and media subgroups that
support the growth of animal cells, plant cells, bacterial cells or
yeast cells. Particularly preferred buffers that may be prepared
according to the invention include balanced salt solutions which
are isotonic for animal cells, plant cells, bacterial cells or
yeast cells.
[0069] Examples of animal cell culture media that may be prepared
according to the present invention include, but are not limited to,
DMEM, RPMI-1640, MCDB 131, MCDB 153, MDEM, IMDM, MEM, M199, McCoys
5A, Williams' Media E, Leibovitz's L-15 Medium, Grace's Insect
Medium, IPL-41 Insect Medium, TC-100 Insect Medium, Schneider's
Drosophila Medium, Wolf & Quimby's Amphibian Culture Medium,
cell-specific serum-free media (SFM) such as those designed to
support the culture of keratinocytes, endothelial cells,
hepatocytes, melanocytes, etc., F10 Nutrient Mixture and F12
Nutrient Mixture. Other media, media supplements and media
subgroups suitable for preparation by the invention are available
commercially (e.g., from Life Technologies, Inc.; Rockville, Md.,
and Sigma; St. Louis, Mo.). Formulations for these media, media
supplements and media subgroups, as well as many other commonly
used animal cell culture media, media supplements and media
subgroups are well-known in the art and may be found, for example
in the GIBCO/BRL Catalogue and Reference Guide (Life Technologies,
Inc.; Rockville, Md.) and in the Sigma Animal Cell Catalogue
(Sigma; St. Louis, Mo.).
[0070] Examples of plant cell culture media that may be prepared
according to the present invention include, but are not limited to,
Anderson's Plant Culture Media, CLC Basal Media, Gamborg's Media,
Guillard's Marine Plant Culture Media, Provasoli's Marine Media,
Kao and Michayluk's Media, Murashige and Skoog Media, McCown's
Woody Plant Media, Knudson Orchid Media, Lindemann Orchid Media,
and Vacin and Went Media. Formulations for these media, which are
commercially available, as well as for many other commonly used
plant cell culture media, are well-known in the art and may be
found for example in the Sigma Plant Cell Culture Catalogue (Sigma;
St. Louis, Mo.).
[0071] Examples of bacterial cell culture media that may be
prepared according to the present invention include, but are not
limited to, Trypticase Soy Media, Brain Heart Infusion Media, Yeast
Extract Media, Peptone-Yeast Extract Media, BeefInfusion Media,
Thioglycollate Media, Indole-Nitrate Media, MR-VP Media, Simmons'
Citrate Media, CTA Media, Bile Esculin Media, Bordet-Gengou Media,
Charcoal Yeast Extract (CYE) Media, Mannitol-salt Media,
MacConkey's Media, Eosin-methylene blue (EMB) media, Thayer-Martin
Media, Salmonella-Shigella Media, and Urease Media. Formulations
for these media, which are commercially available, as well as for
many other commonly used bacterial cell culture media, are
well-known in the art and may be found for example in the DIFCO
Manual (DIFCO; Norwood, Mass.) and in the Manual of Clinical
Microbiology (American Society for Microbiology, Washington,
D.C.).
[0072] Examples of fungal cell culture media, particularly yeast
cell culture media, that may be prepared according to the present
invention include, but are not limited to, Sabouraud Media and
Yeast Morphology Media (YMA). Formulations for these media, which
are commercially available, as well as for many other commonly used
yeast cell culture media, are well-known in the art and may be
found for example in the DIFCO Manual (DIFCO; Norwood, Mass.) and
in the Manual of Clinical Microbiology (American Society for
Microbiology, Washington, D.C.).
[0073] As the skilled artisan will appreciate, any of the above
media of the invention may also include one or more additional
components, such as indicating or selection agents (e.g., dyes,
antibiotics, amino acids, enzymes, substrates and the like),
filters (e.g., charcoal), salts, polysaccharides, ions, detergents,
stabilizers, and the like.
[0074] In a particularly preferred embodiment of the invention, the
above-described culture media may comprise one or more buffer
salts, preferably sodium bicarbonate, at concentrations sufficient
to provide optimal buffering capacity for the culture medium.
According to one aspect of the invention, a buffer salt, such as
sodium bicarbonate, may be added in powdered form to the powdered
medium prior to, during or following agglomeration of the medium.
In one example of this aspect of the invention, the sodium
bicarbonate may be added to the culture medium prior to, during or
following agglomeration with an appropriate solvent (such as water,
serum or a pH-adjusting agent such as an acid (e.g., HCl at a
concentration of 1M to 5M, preferably at 1M) or a base (e.g., NaOH
at a concentration of 1M to 5M, preferably at 1M) such that, upon
reconstitution of the agglomerated medium the culture medium is at
the optimal or substantially optimal pH for cultivation of a
variety of cell types. For example, bacterial cell culture media
prepared by the present methods will, upon reconstitution,
preferably have a pH of about 4-10, more preferably about 5-9 or
about 6-8.5. fungal (e.g., yeast) cell culture media prepared by
the present methods will, upon reconstitution, preferably have a pH
of about 3-8, more preferably about 4-8 or about 4-7.5; animal cell
culture media prepared by the present methods will, upon
reconstitution, preferably have a pH of about 6-8 or about 7-8,
more preferably about 7-7.5 or about 7.2-7.4; and plant cell
culture media prepared by the present methods will, upon
reconstitution, preferably have a pH of about 4-8, preferably about
4.5-7, 5-6 or 5.5-6. Of course, optimal pH for a given culture
medium to be used on a particular cell type may also be determined
empirically by one of ordinary skill using art-known methods.
[0075] In another example, one or more buffer salts, e.g., sodium
bicarbonate, may be added directly to a powdered nutritive medium
by agglomerating the buffer(s) into the medium using a fluid bed
apparatus, or by spray-drying the buffer(s) onto a dry or
agglomerated powdered medium (using a spray-drying apparatus as
described below). In a related aspect, a pH-adjusting agent such as
an acid (e.g., HCl) or a base (e.g., NaOH) may be added to a
powdered nutritive medium, which may contain one or more buffer
salts (such as sodium bicarbonate), by agglomeration of the
pH-adjusting agent into the powdered nutritive medium in a fluid
bed apparatus, by spray-drying the pH-adjusting agent onto the
powdered or agglomerated nutritive medium, or by a combination
thereof; this approach obviates the subsequent addition of a
pH-adjusting agent after reconstitution of the powdered medium.
Thus, the invention provides a powdered nutritive culture medium
useful in cultivation or growth of cells in vitro that, upon
reconstitution with a solvent (e.g., water or serum), has a pH that
is optimal for the support of cell cultivation or growth without a
need for adjustment of the pH of the liquid medium. This type of
medium, defined herein as "automatically pH-adjusting medium,"
therefore obviates the time-consuming and error-prone steps of
adding buffer(s) to the medium after reconstitution and adjusting
the pH of the medium after dissolution of the buffer(s). For
example, a mammalian cell culture medium prepared according to
these methods may, upon reconstitution, have a pH of between about
7.1 to about 7.5, more preferably between about 7.1 to about 7.4,
and most preferably about 7.2 to about 7.4 or about 7.2 to about
7.3. The preparation of one example of such an automatically
pH-adjusting culture medium is shown in more detail below in
Examples 3 and 6.
[0076] Examples of media supplements that may be prepared as
powders by the present methods include, without limitation, animal
sera (such as bovine sera (e.g., fetal bovine, newborn calf and
calf sera), human sera, equine sera, porcine sera, monkey sera, ape
sera, rat sera, murine sera, rabbit sera, ovine sera and the like),
defined replacements such as LipoMAX.RTM., OptiMAb.RTM.,
Knock-Out.TM. SR (each available from Life Technologies, Inc.,
Rockville, Md.), hormones (including steroid hormones such as
corticosteroids, estrogens, androgens (e.g., testosterone) and
peptide hormones such as insulin, cytokines (including growth
factors (e.g., EGF, aFGF, bFGF, HGF, IGF-1, IGF-2, NGF and the
like), interleukins, colony-stimulating factors, interferons and
the like), neurotransmitters, lipids (including phospholipids,
sphingolipids, fatty acids, cholesterol and the like), attachment
factors (including extracellular matrix components such as
fibronectin, vitronectin, laminins, collagens, proteoglycans,
glycosaminoglycans and the like), and extracts of animal tissues,
organs or glands (such as bovine pituitary extract, bovine brain
extract, chick embryo extract, bovine embryo extract, chicken meat
extract, achilles tendon and extracts thereof) and the like). Other
media supplements that may be produced by the present methods
include a variety of proteins (such as serum albumins, particularly
bovine or human serum albumins; immunoglobulins and fragments or
complexes thereof; aprotinin; hemoglobin; haemin or haematin;
enzymes (such as trypsin, collagenases, pancreatinin or dispase);
lipoproteins; fetuin; ferritin; etc.), which may be natural or
recombinant; vitamins; amino acids and variants thereof (including,
but not limited to, L-glutamine and cystine), enzyme co-factors;
polysaccharides; salts or ions (including trace elements such as
salts or ions of molybdenum, vanadium, cobalt, manganese, selenium,
and the like); and other supplements and compositions that are
useful in cultivating cells in vitro that will be familiar to one
of ordinary skill. These sera and other media supplements are
available commercially (for example, from Life Technologies, Inc.,
Rockville, Md., and Sigma Cell Culture, St. Louis, Mo.);
alternatively, sera and other media supplements described above may
be isolated from their natural sources or produced recombinantly by
art-known methods that will be routine to one of ordinary skill
(see Freshney, R. I., Culture of Animal Cells, New York: Alan R.
Liss, Inc., pp. 74-78 (1983), and references cited therein; see
also Harlow, E., and Lane, D., Antibodies: A Laboratory Manual,
Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, pp.
1145-120 (1988)).
[0077] Examples of buffers that may be prepared according to the
present invention include, but are not limited to,
phosphate-buffered saline (PBS) formulations, Tris-buffered saline
(TBS) formulations, HEPES-buffered saline (HBS) formulations,
Hanks' Balanced Salt Solutions (HBSS), Dulbecco's PBS (DPBS),
Earle's Balanced Salt Solutions, Puck's Saline Solutions, Murashige
and Skoog Plant Basal Salt Solutions, Keller's Marine Plant Basal
Salt Solutions, Provasoli's Marine Plant Basal Salt Solutions, and
Kao and Michayluk's Basal Salt Solutions. Formulations for these
buffers, which are commercially available, as well as for many
other commonly used buffers, are well-known in the art and may be
found for example in the GIBCO/BRL Catalogue and Reference Guide
(Life Technologies, Inc.; Rockville, Md.), in the DIFCO Manual
(DIFCO; Norwood, Mass.), and in the Sigma Cell Culture Catalogues
for animal and plant cell culture (Sigma; St. Louis, Mo.).
Preparation of Powdered Media, Media Supplements, Media Subgroups
and Buffers
[0078] The methods of the present invention provide for the
preparation of the above-described powdered nutritive media, media
supplements, media subgroups and buffers. These powdered media,
supplements, subgroups and buffers are preferably prepared using
fluid bed technology (i.e., "agglomeration") and/or via
spray-drying.
[0079] In one aspect of the invention, the powdered nutritive
media, media supplements, media subgroups and buffers are prepared
using fluid bed technology to agglomerate the solutions of media,
media supplements, media subgroups or buffers, thereby producing
their dry powdered forms. Fluid bed technology is a process of
producing agglomerated powders having altered characteristics
(particularly, for example, solubility) from the starting
materials. In general applications of the technology, powders are
suspended in an upwardly moving column of air while at the same
time a controlled and defined amount of liquid is injected into the
powder stream to produce a moistened state of the powder; mild heat
is then used to dry the material, producing an agglomerated
powder.
[0080] Apparatuses for producing and/or processing particulate
materials by fluid bed technology are available commercially (e.g.,
from Niro, Inc./Aeromatic-Fielder; Columbia, Md.), and are
described, for example, in U.S. Pat. Nos. 3,771,237; 4,885,848;
5,133,137; 5,357,688; and 5,392,531; and in WO 95/13867; the
disclosures of all of the foregoing patents and applications are
incorporated by reference herein in their entireties. Such
apparatuses have been used to prepare agglomerated powders of
various materials, including milk whey (U.S. Pat. No. 5,006,204),
acidulated meat emulsions (U.S. Pat. No. 4,511,592), proteases
(U.S. Pat. No. 4,689,297) and other proteins (DK 167090 B1), and
sodium bicarbonate (U.S. Pat. No. 5,325,606).
[0081] According to this aspect of the invention, fluid bed
technology may be used to prepare bulk agglomerated nutritive
media, media supplements, media subgroups and buffers. In the
practice of this aspect of the invention, a dry powdered nutritive
medium, medium supplement or buffer is placed into a fluid bed
apparatus and is subjected to agglomeration therein. Powdered
nutritive media (particularly powdered cell culture media),
powdered media supplements (particularly powdered animal sera) and
powdered buffers (particularly powdered buffered salines), may be
obtained pre-made from commercial sources (e.g., Life Technologies,
Inc.; Rockville, Md.). Alternatively, powdered nutritive media,
media supplements, media subgroups or buffers may be made by
admixing individual components or sets of components according to
the formulations described above. Such formulations may include
components which typically are not present in powdered nutritive
media, media supplement, media subgroup and buffer formulations due
to their instability, such as serum, L-glutamine, cystine, insulin,
transferrin, lipids (particularly phospholipids, sphingolipids,
fatty acids and cholesterol), cytokines (particularly growth
factors, interleukins, colony-stimulating factors and interferons),
neurotransmitters and buffers (particularly sodium bicarbonate). If
L-glutamine is added to the formulation, it may be in the form of a
complex with divalent cations such as calcium or magnesium (see
U.S. Pat. No. 5,474,931). In another example, two or more powdered
components may be admixed and then agglomerated to produce complex
media, media supplements, media subgroups or buffers. For example,
a powdered nutritive medium may be mixed with a powdered serum
(produced, for example, by spray-drying as described below) such as
FBS at a serum concentration of about 0.1%, 0.2%, 0.5%, 1%, 2%,
2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 50% or higher (w/w as a
percentage of the powdered medium); the resulting powdered
medium-serum mixture may then be agglomerated to produce an
agglomerated medium-serum complex that will readily dissolve in a
reconstituting solvent and thus be ready for use without further
supplementation.
[0082] Once the powdered nutritive media, media supplement, media
subgroup or buffer (or mixture thereof) is placed into the fluid
bed apparatus, it is subjected to suspension in an upwardly moving
column of a gas, preferably atmospheric air or an inert gas such as
nitrogen, and is passed through one or more particle filters. Since
most dry powder, non-agglomerated nutritive media, media
supplements, media subgroups and buffers are of a relatively small
particle size, filters to be used in the invention should be mesh
screens that allow air to flow through but that retain the powders,
for example filters of about 1-100 mesh, preferably about 2-50
mesh, more preferably about 2.5-35 mesh, still more preferably
about 3-20 mesh or about 3.5-15 mesh, and most preferably about 4-6
mesh.
[0083] After placement within the fluid bed chamber, the dry powder
nutritive media, media supplement, media subgroup or buffer (or
mixture thereof) is then subjected to agglomeration by injecting,
preferably using a spray nozzle on the fluid bed apparatus, a
defined and controlled amount of solvent into the powder, to
produce a moistened powder. Preferred solvents for use in the
present invention are any solvent that is compatible with the
formulation of the nutritive media, media supplement, media
subgroup or buffer. By "compatible" is meant that the solvent does
not induce irreversible deleterious changes in the physical or
performance characteristics of the nutritive media, media
supplement, media subgroup or buffer, such as breakdown or
aggregation of the nutrient components of the nutritive medium or
changes in the ionic characteristics of the buffer. Particularly
preferred solvents for use in the invention are water (most
particularly distilled and/or deionized water), serum (particularly
bovine or human serum and most particularly fetal bovine serum or
calf serum), organic solvents (particularly dimethylsulfoxide,
acetone, ethanol and the like), any of which may contain one or
more additional components (e.g., salts, polysaccharides, ions,
detergents, stabilizers, etc.).
[0084] In some aspects of the invention, it may be desirable or
advantageous to include in the solvent one or more ingredients
that, due to the concentrations of the components required in the
final product, cannot be optimally incorporated into the product by
other methods such as ball-milling. In one such aspect, the
component may be dissolved, suspended, colloided or otherwise
introduced into the solvent at the desired concentration, prior to
use of the solvent in agglomeration of the powdered media, media
supplement, media subgroup or buffer of the invention. Components
that may be advantageously incorporated into the solvent in
accordance with this aspect of the invention include, but are not
limited to, one or more of the above-described sera, hormones,
cytokines, neurotransmitters, lipids, attachment factors, proteins,
amino acids, vitamins, enzyme cofactors, polysaccharides, salts,
ions, buffers and the like.
[0085] The solvent should be introduced into the dry powder in a
volume that is dependent upon the mass of powdered media, media
supplement, media subgroup or buffer to be agglomerated. Preferred
volumes of solvent per 500 grams of nutritive media, media
supplement, media subgroup or buffer are about 5-100 ml, more
preferably about 10-50 ml, still more preferably about 25-50 ml,
and most preferably about 35 ml. Preferred solvent introduction
rates per 500 grams of nutritive media, media supplement, media
subgroup or buffer are a rate of about 1-10 ml/min, preferably
about 2-8 ml/min, more preferably about 4-8 ml/min acid most
preferably about 6 ml/min. In some situations, it may be desirable
to cycle between adding solvent for about one minute and then not
adding solvent for about one minute (allowing drying of the powder
within the apparatus chamber), so as to prevent dumping of the
powder during agglomeration.
[0086] Once agglomeration of the powder is complete, as evidenced
by a larger particle size than that of the original, unagglomerated
powder and by the absence of fine dust particles in the
agglomerated powder, the powder is thoroughly dried in the
apparatus. Preferred apparatus temperatures for drying of the
agglomerated powder are about 50-80.degree. C., more preferably
about 55-75.degree. C., and most preferably about 60-65.degree. C.;
powder is preferably dried in the apparatus for about 3-10 minutes
and most preferably for about 5-7 minutes, per 500 grams of
powder.
[0087] In another aspect of the invention, powdered nutritive
media, media supplements, media subgroups and buffers may be
prepared by spray-drying. In this aspect of the invention, the
nutritive media, media supplements, media subgroups and buffers in
their liquid forms are placed into a spray-drying apparatus; these
liquids are then converted into their corresponding powders by
spraying the solution into a chamber in the apparatus under
appropriate conditions to produce the powders, such as under
controlled temperature and humidity, until the powders are formed.
In some situations, it may be desirable or advantageous to
spray-dry complex mixtures of two or more of the above-described
media, media supplements, media subgroups and/or buffers. For
example, liquid nutritive media containing animal sera at a desired
concentration, or liquid animal sera containing nutritive media
components at desired concentrations, may be mixed and then
prepared as spray-dried powders according to the methods of the
invention.
[0088] In a typical spray-drying approach, the liquid nutritive
media, media supplements, media subgroups and buffers are aspirated
into the apparatus and are atomized into a spray with a rotary- or
nozzle-type atomizer. The resulting atomized liquid spray is then
mixed with a gas (e.g., nitrogen or more preferably air) and
sprayed into a drying chamber under conditions sufficient to
promote production of a powdered product. In a preferred aspect of
the invention, these conditions may comprise electronic control of
the temperature and humidity within the chamber such that final
drying of the product is promoted. Under these conditions, the
solvent in the liquid evaporates in a controlled manner, thereby
forming free-flowing particles (i.e., powder) of the nutritive
media, media supplements, media subgroups or buffers of the
invention. The powder is then discharged from the drying chamber,
passed through one or more filters (such as the mesh screens
described above for fluid bed preparation) and collected for
further processing (e.g., packaging, sterilization, etc.). In some
applications, particularly when producing powders from
heat-sensitive formulations of nutritive media, media supplements,
media subgroups and buffers, the spray-drying apparatus may be
combined with a fluid bed apparatus integrated within the drying
chamber, which allows the introduction of agglomerating solvents
such as those described above into the spray-dried powder to
produce agglomerated spray-dried powdered nutritive media, media
supplements, media subgroups and buffers.
[0089] Apparatuses for producing particulate materials from liquid
materials by spray-drying (with or without integrated fluid bed
technology) are available commercially (e.g., from Niro,
Inc./Aeromatic-Fielder, Columbia, Md.), and are described, for
example, in the "Spray Drying," "Powdered Pharmaceuticals by Spray
Drying" and "Fresh Options in Drying" technical brochures of Niro,
Inc./Aeromatic-Fielder, the disclosures of which are incorporated
by reference herein in their entireties. According to this
manufacturer, such apparatuses have been used to prepare powders of
various materials, including dairy products, analgesics,
antibiotics, vaccines, vitamins, yeasts, vegetable protein, eggs,
chemicals, food flavorings and the like. In the present invention,
spray-drying has been found to be particularly useful for the
preparation of powdered media supplements, such as sera and in
particular those sera described above, most particularly human and
bovine sera (such as fetal bovine serum and calf serum).
[0090] In the practice of this aspect of the invention, the liquid
nutritive media, media supplements, media subgroups, buffers or
pH-adjusting agents should be sprayed into the chamber through the
atomizer at a spray rate of about 25-100 g/min, preferably at a
spray rate of about 30-90 g/min, 35-85 g/min, 40-80 g/min, 45-75
g/min, 50-75 g/min, 55-70 g/min, or 60-65 g/min, and more
preferably at about 65 g/min. The inlet air temperature in the
atomizer is preferably set at about 100-300.degree. C., more
preferably at about 150-250.degree. C., and most preferably at
about 200.degree. C., with an outlet temperature of about
50-100.degree. C., more preferably about 60-80.degree. C., and most
preferably about 70.degree. C. Air flow in the atomizer is
preferably set at about 50-100 kg/hr, more preferably about 75-90
kg/hr, and most preferably about 80.0 kg/hr, at a nozzle pressure
of about 1-5 bar, more preferably about 2-3 bar, and most
preferably about 2.0 bar. These conditions and settings have been
found in the present invention to be preferable for production of a
variety of nutritive media, media supplements, media subgroups and
buffer powders by spray-drying, particularly for the production of
the above-described powdered sera. Following drying, the
spray-dried powdered nutritive media, media supplements, media
subgroups or buffers may be collected in the drying chamber through
one or more filters, preferably such as those described above for
fluid bed technology.
[0091] Following this preparation, the powders of the invention
prepared by the above-described fluid bed or spray-drying methods
have altered physical characteristics from the starting powders or
from powdered media, supplements, subgroups and buffers prepared by
lyophilizing the corresponding liquids. For example, non-processed
or lyophilized powders often produce significant dust when used,
and dissolve poorly or slowly in various solvents, while
agglomerated or spray-dried powders are substantially dust-free
and/or dissolve rapidly. Typically, the powdered media, media
supplements, media subgroups and buffers of the invention will
exhibit both reduced dusting and more rapid dissolution than their
powdered counterparts prepared by standard techniques such as
ball-milling. In some powders which are substantially dust-free but
which may not demonstrate enhanced dissolution, the powders may be
rapidly dissolved by rapid mechanical solvation of the powder, such
as using a mechanical impeller, or by first providing a solvent
mist over the powder such as by spray solvation.
[0092] In another aspect of the invention, the spray-drying and
agglomeration approaches described above may be combined to produce
agglomerated spray-dried nutritive media, media supplement, media
subgroup and buffer powders. In this aspect, a powdered medium,
supplement, subgroup or buffer that has been prepared by
spray-drying may, after having been spray-dried, then be
agglomerated with a solvent (such as those described above) to
further improve the performance and physical characteristics of the
resultant medium, supplement, subgroup or buffer. For example, an
animal serum powder may be prepared by spray-drying liquid animal
serum as described above, and this spray-dried serum powder may
then be mixed into dry powder nutritive media (prepared by
spray-drying or by standard techniques such as ball-milling); this
mixed powder may then be agglomerated as described above.
Alternatively, a spray-dried nutritive medium, medium supplement,
medium subgroup or buffer powder may be agglomerated as above, to
improve the dissolution properties of the powder. This approach may
be particularly advantageous when spray-drying liquids with low
(about 1-10%) solids content, such as liquid animal sera. As one of
ordinary skill will appreciate, these approaches will facilitate
preparation of a large batch of one or more components (e.g., sera
or other media supplements) to be used as a stock for addition to a
powdered medium, supplement, subgroup or buffer at a desired
concentration, while also obtaining the above-described benefits of
agglomeration. In addition, this approach may reduce inter-lot
variability which may be a problem with certain media supplements
(particularly animal sera).
[0093] The agglomerated or spray-dried powdered nutritive media,
media supplements, media subgroups or buffers prepared as described
above may then be packaged, for example into containers such as
vials, tubes, bottles, bags, pouches, boxes, cartons, drums and the
like, prior to or following sterilization as described below. In
one such aspect of the invention, the powdered media, media
supplements, media subgroups or buffers may be packaged into a
compact, vacuum-packed form such as that known in the art as a
"brick-pack" wherein the powder is packaged into a flexible
container (such as a bag or a pouch) that is sealed while being
evacuated. Such packages may advantageously comprise one or more
access ports (such as valves, luer-lock ports, etc.) allowing the
introduction of a solvent (e.g., water, sera; media or other
aqueous or organic solvents or solutions) directly into the package
to facilitate rapid dissolution of the powder. In a related aspect,
the package may comprise two or more adjacent compartments, one or
more of which may contain one or more of the dry powder media,
media supplements, media subgroups or buffers of the invention and
one or more other of which may contain one or more aqueous or
organic solvents which may be sterile. In this aspect, the dry
powder may then be dissolved by simply removing or breaking the
barrier between the compartments, ideally without loss of
sterility, to allow admixture of the powder and the solvent such
that the powder dissolves and produces a sterile nutritive medium,
medium supplement, medium subgroup or buffer at a desired
concentration.
[0094] Packaged media, media supplements, media subgroups and
buffers of the invention are preferably stored for the extended
times, and at the temperatures, noted above, typically for about
1-24 months at temperatures of less than about 30.degree. C., more
preferably at temperatures of less than about 20-25.degree. C.,
until use. Unlike traditional powdered media, media supplements,
media subgroups or buffers, storage at reduced temperatures (e.g.,
0-4.degree. C.) is not necessary for the maintenance of performance
characteristics of the media, media supplements, media subgroups
and buffers prepared by the present methods. Of course, other
storage temperatures may be required for those aspects of the
invention where the packages also comprise separate compartments
containing one or more solvents; in these cases, the optimal
storage conditions will be dictated by the storage requirements of
the solvent(s) which will be known to the skilled artisan.
Sterilization and Packaging
[0095] The invention also provides methods for sterilizing the
nutritive media, media supplements, media subgroups and buffers of
the invention, as well as for sterilizing powdered nutritive media,
media supplements, media subgroups and buffers prepared by standard
methods such as ball-milling or lyophilization. Since nutritive
media, media supplements, media subgroups and buffers are usually
prepared in large volume solutions and frequently contain heat
labile components, they are not amenable to sterilization by
irradiation or by heating. Thus, nutritive media, media
supplements, media subgroups and buffers are commonly sterilized by
contaminant-removal methods such as filtration, which significantly
increases the expense and time required to manufacture such media,
media supplements, media subgroups and buffers.
[0096] Powdered nutritive media, media supplements, media subgroups
and buffers prepared according to the methods of the invention
(e.g., by spray-drying of liquid media, media supplements, media
subgroups or buffers, or by agglomeration of powdered media, media
supplements, media subgroups or buffers), or by standard methods
such as ball-milling (of powdered components) or lyophilization (of
liquid forms of the media, supplements, subgroups or buffers),
however, can be sterilized by less expensive and more efficient
methods. For example, powdered nutritive media, media supplements,
media subgroups or buffers (prepared as described above by
spray-drying or lyophilization of a liquid form, or by
agglomeration of a powdered form, of the media, supplements,
subgroups or buffers) may be irradiated under conditions favoring
sterilization of these powders. Preferably, this irradiation is
accomplished in bulk (i.e., following packaging of the nutritive
media, media supplement, media subgroup or buffer), and most
preferably this irradiation is accomplished by exposure of the bulk
packaged media, media supplement, media subgroup or buffer of the
invention to a source of gamma rays under conditions such that
bacteria, fungi, spores or viruses that may be resident in the
powdered media, media supplements, media subgroups or buffers are
inactivated (i.e., prevented from replicating). Alternatively,
irradiation may be accomplished by exposure of the powdered media,
media supplement, media subgroup or buffer, prior to packaging, to
a source of gamma rays or a source of ultraviolet light. The media,
media supplements, media subgroups and buffers of the invention may
alternatively be sterilized by heat treatment (if the subgroups of
the nutritive media, media supplement, media subgroup or buffer are
heat stable), for example by flash pasteurization or autoclaving.
As will be understood by one of ordinary skill in the art, the dose
of irradiation or heat, and the time of exposure, required for
sterilization depend upon the bulk of the materials to be
sterilized.
[0097] In a particularly preferred aspect of the invention, the
bulk powered nutritive media, media supplements, media subgroups or
buffers are exposed to a source of .gamma. irradiation at a total
dosage of about 10-100 kilograys (kGy), preferably a total dosage
of about 15-75 kGy, 15-50 kGy, 15-40 kGy or 20-40 kGy, more
preferably a total dosage of about 20-30 kGy, and most preferably a
total dosage of about 25 kGy, for about 1 hour to about 7 days,
more preferably about 1 hour to about 5 days, 1 hour to about 3
days, about 1-24 hours or about 1-5 hours, and most preferably
about 1-3 hours ("normal dose rate"). Alternatively, the bulk
powders of the invention may be sterilized at a "slow dose rate" of
a total dosage of about 25-100 kGy over a period of about 1-5 days.
During irradiation, the powdered nutritive media, media
supplements, media subgroups or buffers are preferably stored at a
temperature of about -70.degree. C. to about room temperature
(about 20-25.degree. C.), most preferably at about -70.degree. C.
One of ordinary skill will appreciate, of course, that radiation
dose and exposure times may be adjusted depending upon the bulk
and/or mass of material to be irradiated; typical optimal
irradiation dosages, exposure times and storage temperatures
required for sterilization of bulk powdered materials by
irradiation or heat treatment are well-known in the art.
[0098] Following sterilization, unpackaged nutritive media, media
supplements, media subgroups and buffers may be packaged under
aseptic conditions, for example by packaging the media, media
supplements, media subgroups or buffers into containers such as
sterile tubes, vials, bottles, bags, pouches, boxes, cartons, drums
and the like, or in the vacuum packaging or integrated
powder/solvent packaging described above. Sterile packaged media,
media supplements, media subgroups and buffers may then be stored
for extended periods of time as described above.
Use of the Nutritive Media, Media Supplements, Media Subgroups and
Buffers
[0099] The present invention thus provides powdered nutritive
media, media supplements, media subgroups and buffers that are
readily soluble in a rehydrating solvent and that are substantially
dust free. For use, the agglomerated or spray-dried media, media
supplement, media subgroup or buffer may be hydrated (or
"reconstituted") in a volume of a solvent sufficient to produce the
desired nutrient, electrolyte, ionic and pH conditions required for
the particular use of the solvated media, media supplement, media
subgroup or buffer. This reconstitution is particularly facilitated
in the present invention, since the present media, media
supplements, media subgroups and buffers will rapidly go into
solution and will produce little if any dust or insoluble material,
unlike lyophilized or ball-milled nutritive media, media
supplements, media subgroups or buffers.
[0100] Preferred solvents for use in reconstituting the powdered
nutritive media, media supplements, media subgroups and buffers of
the invention include, but are not limited to, water (most
particularly distilled and/or deionized water), serum (particularly
bovine or human serum and most particularly fetal bovine serum or
calf serum), organic solvents (particularly dimethylsulfoxide,
acetone, ethanol and the like), or any combination thereof, any of
which may contain one or more additional components (e.g., salts,
polysaccharides, ions, detergents, stabilizers, etc.). For example,
powdered media supplements (such as animal sera) and buffers are
preferably reconstituted in water to a 1.times. final
concentration, or optionally to a higher concentration (e.g.,
2.times., 2.5.times., 5.times., 10.times., 20.times., 25.times.,
50.times., 100.times., 500.times., 1000.times., etc.) for the
preparation of stock solutions or for storage. Alternatively,
powdered culture media may be reconstituted in a solution of media
supplements (e.g., sera such as FBS) in water, such as those
solutions wherein the media supplement is present at a
concentration, for example, of 0.5%, 1%, 2%, 2.5%, 5%, 7.5%, 10%,
15%, 20%, 25%, 50%, or higher, vol/vol in the water.
[0101] Reconstitution of the powdered nutritive media, media
supplements, media subgroups or buffers is preferably accomplished
under aseptic conditions to maintain the sterility of the
reconstituted media, media supplement, media subgroup or buffer,
although the reconstituted media, media supplement, media subgroup
or buffer may alternatively be sterilized, preferably by filtration
or other sterilization methods that are well-known in the art,
following rehydration. Following their reconstitution, media, media
supplements, media subgroups and buffers should be stored at
temperatures below about 10.degree. C., preferably at temperatures
of about 0-4.degree. C., until use.
[0102] The reconstituted nutritive media, media supplements, media
subgroups and buffers may be used to culture cells according to
standard cell culture techniques which are well-known to one of
ordinary skill in the art. In such techniques, the cells to be
cultured are contacted with the reconstituted media, media
supplement, media subgroup or buffer of the invention under
conditions favoring the cultivation of the cells (such as
controlled temperature, humidity, lighting and atmospheric
conditions). Cells which are particularly amenable to cultivation
by such methods include, but are not limited to, bacterial cells,
yeast cells, plant cells and animal cells. Such bacterial cells,
yeast cells, plant cells and animal cells are available
commercially from known culture depositories, e.g., American Type
Culture Collection (Rockville, Md.), Invitrogen (La Jolla, Calif.)
and others that will be familiar to one of ordinary skill in the
art. Preferred animal cells for cultivation by these methods
include, but are not limited to, insect cells (most preferably
Drosophila cells, Spodoptera cells and Trichoplusa cells), nematode
cells (most preferably C. elegans cells) and mammalian cells
(including but not limited to CHO cells, COS cells, VERO cells, BHK
cells, AE-1 cells, SP2/0 cells, L5.1 cells, hybridoma cells and
most preferably human cells such as 293 cells, PER-C6 cells and
HeLa cells), any of which may be a somatic cell, a germ cell, a
normal cell, a diseased cell, a transformed cell, a mutant cell, a
stem cell, a precursor cell or an embryonic cell, and any of which
may be an anchorage-dependent or anchorage-independent (i.e.,
"suspension") cell.
Cells
[0103] In another aspect, the invention relates to methods for
producing dry cell powder compositions comprising one or more
cells, and to dry cell powders produced by these methods. These
methods thus produce cell-containing compositions wherein the cells
are preserved and may be stored for extended periods of time until
use. In this way, the methods of the invention overcome some of the
drawbacks of traditional methods of cell preservation (e.g.,
freezing) such as the need for cyropreservation equipment and the
use of certain cryopreservatives that may be toxic to the
cells.
[0104] Methods according to this aspect of the invention may
comprise one or more steps. For example, one such method may
comprise obtaining one or more cells to be dried, forming an
aqueous cell suspension by suspending the one or more cells in an
aqueous solution, and spray-drying the cell suspension under
conditions favoring the production of a dried powder. These methods
may further comprise contacting the one or more cells with one or
more stabilizing or preserving compounds (e.g., a polysaccharide,
including but not limited to trehalose). The aqueous solution used
to form the cell suspension preferably comprises one or more
components, such as one or more of the above-described nutritive
media, media supplements, media subgroups, salts or buffers.
Preferably, the aqueous solution used to form the cell suspension
is adjusted to optimal or substantially optimal tonicity and
osmolality for the cell type being dried. The aqueous solution may
optionally comprise one or more additional components, such as one
or more polysaccharides, ions, detergents, stabilizing or
preserving compounds (including trehalose), and the like. In
aspects of the invention wherein the one or more cells are
contacted with one or more stabilizing or preserving compounds, the
stabilizing or preserving compounds may be incorporated into the
aqueous solution used to form the aqueous cell suspension.
Alternatively, the stabilizing or preserving compounds may be
sprayed or agglomerated onto the dry cell powder after formation of
the powder.
[0105] Once the dry cell powder has been formed by the
above-described methods, the powder may optionally be agglomerated
with a solvent according to methods described above for
agglomeration of dry powders. Any solvent that is compatible with
the cell type being dried may be used to agglomerate the dry cell
powder, including but not limited to water, a nutritive medium
solution, a nutritive medium supplement solution (including sera,
particularly bovine sera (most particularly fetal bovine and calf
sera) and human sera), a buffer solution, a salt solution, and
combinations thereof.
[0106] A variety of cells may be dried according to the methods of
the invention, including prokaryotic (e.g., bacterial) and
eukaryotic (e.g., fungal (especially yeast), animal (especially
mammalian, including human) and plant) cells, particularly those
cells, tissues, organs, organ systems, and organisms described
above. Once the dried cells have been produced, they may be
packaged aseptically and stored for extended periods of time (e.g.,
several months to several years), preferably at temperatures of
about 0-30.degree. C., 4-25.degree. C., 10-25.degree. C., or
20-25.degree. C. (i.e., "room temperature") until use. For use in
preparing cultures of viable cells, the dry cell powder may be
aseptically reconstituted, into a cell suspension comprising one or
more viable cells, with an aqueous solvent (e.g., sterile water,
buffer solutions, media supplements, culture media, or combinations
thereof) and cultured according to standard art-known protocols.
Alternatively, the dry cell powder may be reconstituted into a cell
suspension where cell viability is not essential, for example for
preparation of an immunogen to be used for immunization of an
animal. In such cases, the dry cell powder may be reconstituted
with any solvent that is compatible with standard immunization
protocols, such as aqueous or organic solvents that may comprise
one or more detergents, adjuvants, etc.
Kits
[0107] The dry powder media, media supplements, media subgroups,
buffers and cells provided by the invention are ideally suited for
preparation of kits. Such a kit may comprise one or more containers
such as vials, test tubes, bottles, packages, pouches, drums, and
the like. Each of the containers may contain one or more of the
above-described nutritive media, media supplements, media subgroups
or buffers of the invention, or combinations thereof. Such
nutritive media, media supplements, media subgroups or buffers may
be hydrated or dehydrated but are typically dehydrated preparations
produced by the methods of the invention. Such preparations may,
according to the invention, be sterile.
[0108] A first container may contain, for example, a nutritive
media, media supplement, media subgroup or a buffer of the
invention, or any component or subgroup thereof, such as any of
those nutritive media, media supplements, media subgroups or
buffers of the invention that are described above. Additional
nutritive media, buffers, extracts, supplements, components or
subgroups may be contained in additional containers in the present
kits. The kits may also contain, in one or more additional
containers, one or more cells such as the above-described bacterial
cells, yeast cells, plant cells or animal cells. Such cells may be
lyophilized, dried, frozen or otherwise preserved, or may be
spray-dried according to the methods of the invention. In addition,
the kits of the invention may further comprise one or more
additional containers, containing, for example, L-glutamine,
optionally complexed with one or more divalent cations (see U.S.
Pat. No. 5,474,931). The kits may further comprise one or more
additional containers containing a solvent to be used in
reconstituting the dry powder nutritive media, media supplements,
media subgroups and/or buffers; such solvents may be aqueous
(including buffer solutions, saline solutions, nutritive medium
solutions, nutritive medium supplement solutions (including sera
such as bovine sera (particularly fetal bovine sera or calf sera)
or human sera), or combinations thereof) or organic. Other
ingredients that are not compatible for admixture with the
nutritive media, buffers, extracts, supplements, components or
subgroups of the invention may be contained in one or more
additional containers to avoid mixing of incompatible
components.
[0109] The number and types of containers contained in a given kit
for making a nutritive media, media supplement, media subgroup or
buffer may vary depending on the type of media, media supplement,
media subgroup or buffer to be prepared. Typically, the kit will
contain the respective containers containing the components or
supplements necessary to make a particular media, media supplement,
media subgroup or buffer. However, additional containers may be
included in the kit of the invention so that different media, media
supplements, media subgroups or buffers can be prepared by mixing
different amounts of various components, supplements, subgroups,
buffers, solvents, etc., to make different media, media supplement,
media subgroup or buffer formulations.
Advantages
[0110] Unexpectedly, the present invention provides for the
preparation of nutritive media, media supplements, media subgroups,
buffers and cells at reduced cost. The cost reductions are due to
the several factors. For example, the media, media supplement,
media subgroup and buffer formulations of the present invention may
be produced with much smaller production facilities since the large
stir tanks required for 1.times. formulations are not required. In
addition, the media, media supplement, media subgroup and buffer
formulations of the present invention may be prepared on an as
needed basis using "just in time" production techniques which
reduce inventory, storage and labor costs. The time required for
the preparation and shipping of the media, media supplement, media
subgroup and buffer formulations may be reduced from 6-8 weeks to
as little as one day. The automatically pH-adjusting media of the
invention also provide significant cost and time savings, and
reduce the tendency for introduction of contamination into
reconstituted media that may occur during the pH adjustment process
according to standard methods using traditional dry powder or bulk
liquid media. The present invention also allows for the preparation
of components of nutritive media, media supplements, media
subgroups or buffers which may be used to prepare very large
quantities of 1.times. media, media supplements, media subgroups or
buffers (e.g., 100,000 liters or more) which would require only one
quality control test compared to multiple quality control tests for
multiple batches produced according to other commonly used
techniques. Importantly, the media, media supplement, media
subgroup and buffer formulations of the present invention are more
consistent between batches since the individual components are more
stable. The dried cell powders of the invention are also
technologically and economically advantageous, since the cells may
be stored, in low volume, for extended periods of time with little
need for specialized equipment beyond that typically available in
the laboratory. In addition, the cells prepared by the present
methods are preserved without being exposed to cryopreservative
reagents which may be toxic to the cells.
[0111] It will be readily apparent to one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein are obvious and may
be made without departing from the scope of the invention or any
embodiment thereof. Having now described the present invention in
detail, the same will be more clearly understood by reference to
the following examples, which are included herewith for purposes of
illustration only and are not intended to be limiting of the
invention.
Example 1
Agglomeration of Typical Dry Powder Media (DPM)
[0112] 1. With a benchtop laboratory fluid bed apparatus (Stera-1;
Niro, Inc./Aeromatic-Fielder; Columbia, Md.): Place 100-500 g of
DPM within the chamber. Place onto apparatus and use the lever to
seal the unit.
[0113] 2. Start the airflow to fluidize (levitate) the DPM. Since
traditional DPM is of relatively fine particle size, setting 4-6
will be needed. Turn on the vacuum device to catch fine DPM
particles, passing through the upper filters. Make sure that the
fluidized powder is approximately central within the chamber with
respect to the lower mesh screen and the upper filters.
[0114] 3. Start the injection device (spray unit) by first plugging
in the compressed air line and then by starting the pump which is
connected to a water source. The goal is to admit .about.6 ml of
water per minute (the flow rate for any given pump based upon RPM
and tubing diameter must be known). In order to prevent clumping of
DPM, alternatively add water for .about.1 minute and then stop for
.about.1 minute, allowing drying to occur in the chamber.
[0115] 4. If filters become coated with DPM during the run so that
blowback does not dislodge powder, turn fan speed down to setting
2-3 until all filters have been blown clear. Then increase running
fan speed to previous level.
[0116] 5. Agglomeration will be complete when .about.35 ml of water
has been added for each 500 g of DPM. This volume will vary
depending upon the DPM formulation. A downward flow of relatively
large agglomerated granules will be seen in the chamber (bottom)
toward the end of the run. Visibly larger particles and absence of
fine dust indicates that the process is complete.
[0117] 6. Allow agglomerated DPM to dry thoroughly for 5-7
minutes.
[0118] 7. At end of run, blow off filters 4 times.
[0119] 8. Turn unit off, disconnect water tube and collect
agglomerated DPM into an airtight container.
[0120] These approaches should be adjusted when using a
process-scale or production-scale fluid bed apparatus. For example,
when the MP-1 (Niro, Inc./Aeromatic-Fielder; Columbia, Md.)
apparatus is used, the following protocol has yielded satisfactory
results: [0121] 1. Seal unit (inflate gaskets). [0122] 2. Start fan
for pre-heat. [0123] 3. Stop fan when inlet air temperature equals
set point. [0124] 4. Deflate gaskets, load material, inflate
gaskets. Steps 5-8 should all be accomplished within one minute:
[0125] 5. Start batch. [0126] 6. Start fan, and turn on filter
cleaning. [0127] 7. Set nozzle atomizing air pressure % output
(check nozzle for vacuum). [0128] 8. Connect liquid feed line.
[0129] 9. Start pump on screen and at pump. [0130] 10. Reset batch
time. [0131] 11. Spray all liquid at set rate (26 g/min). Use
.about.250 ml water for 2 kg powder. [0132] 12. Stop pump at pump
and on screen when all liquid is added. [0133] 13. Reduce airflow
to drying value (for example from 100 to 60). [0134] 14. When
product reaches desired temperature (.about.40.degree. C.), go to
"initial set up" screen and set "batch duration" for a value of 2-3
minutes greater than the present "batch time". [0135] 15. Stop
batch. [0136] 16. Deflate gaskets.
[0137] Typical instrument settings (for bench-, process- and
production-scale apparatuses):
[0138] Drying temperature: 60-65.degree. C.
[0139] Outlet air temperature: .about.33.degree. C.
[0140] Blow out pressure: 5 bar
[0141] Atomizing pressure: 1.5-2.0 bar
[0142] Blow back dwell: 1 after spraying, 2 while spraying
[0143] Capacity of fan: 5 at start of run, 6 after agglomeration is
evident
[0144] Magnahelics: Filter resistance 150-250, Resistance of
perforated control plate .about.50, Air volume: less than 50.
Example 2
Addition of Sodium Bicarbonate as an Integral Part of DPM
[0145] As noted above, sodium bicarbonate is not typically added to
DPM during manufacturing by ball-milling or lyophilization, due to
potential off-gassing and buffering capacity complications
encountered upon storage of the powdered media. This standard
production process thus necessitates the addition of sodium
bicarbonate, and pH adjustment, upon reconstitution of the media.
With the present methods, however, these additional steps may be
obviated by adding the sodium bicarbonate (or any buffering salt)
directly to the powdered medium during manufacturing.
[0146] There are two ways of including sodium bicarbonate (or any
buffering salt) within the DPM: (a) via the injection device and
(b) as part of the DPM.
(a) Injection Device
[0147] Because of the solubility of sodium bicarbonate and the
amounts that generally need to be added to a typical mammalian cell
culture medium, fairly large volumes of liquid would need to be
injected into the powder (significantly greater than the 35 ml of
water mentioned above). This is still possible and in fact may be
preferable if adding another component that similarly requires a
relatively large volume of liquid in order to be added to the DPM,
as is the case with serum for example. In this case, care must be
taken to sequentially add liquid, let dry etc. a number of times to
insure that the DPM does not become clumped within the device.
Using the 6 ml per minute for .about.1 minute and then allowing
drying for another 2 minutes is about right.
[0148] The amount of liquid to add is determined as follows:
Prepare sodium bicarbonate at 75 g/L in water. Example: 250 g of
DPM in the chamber to be agglomerated. Assume 10.0 g of DPM is
required for 1 L of 1.times. liquid medium. Therefore, 250 g
represents 25 L of 1.times. liquid medium. For each L of liquid,
assume (for example) a requirement of 2 g of sodium bicarbonate.
This means that 50 g of bicarbonate is needed. Now, since the
bicarbonate solution is at 75 g/L, then 0.67 L of bicarbonate
solution must be added to the 250 g of DPM.
[0149] The sodium bicarbonate solution would be added similarly to
the process for "agglomeration of a typical DPM" above except that
a longer drying time between cycles is needed since the pH of the
sodium bicarbonate solution is .about.8.00 which can degrade media
components. It is important that the powder never become "soaked"
by addition of bicarbonate solution too rapidly without allowing
sufficient time for thorough drying of the bicarbonate powder
between cycles. Also, longer fluid drying times are required since
it is important to have as low a final moisture content as possible
since moisture would result in liberation of carbon dioxide gas
resulting in loss of buffering capacity and "pillow" formation if
powder is in a foil packet.
(b) As Part of the DPM
[0150] Sodium bicarbonate can be milled into the DPM in a similar
fashion as for other media components prior to fluid bed treatment.
However, in the milling process, the bicarbonate should be added as
the final component. All of the other media components should be
milled as usual and then the mill stopped and the bicarbonate added
last, with further milling to reach proper sized particles. It is
important that all post-milling processing (placement into
containers, etc.) be done in a humidity-controlled environment set
as low as operationally possible (.about.20-40%. Fluid bed
processing should then be performed as soon as possible after
milling. (If not processed the same day, DPM must be double wrapped
and placed within a sealed container with moisture absorbents.)
[0151] The fluid bed process itself is done similarly to the
example given above (with use of 35 ml per 500 g of DPM) except
that drying times after water injection (.about.6 ml/min) should
again be extended: 1 min of injection of water and 2 minutes drying
cycles. It will be noted that the color of the DPM will be deep
red-light purple due to presence of phenol red. Since the DPM has
essentially no moisture content, this does not represent a
degradative situation, and is why fluid bed processing is
essential.
Example 3
DPM that Includes Buffering Salts (e.g., Sodium Bicarbonate) and is
Formulated so that pH of Reconstituted (1.times.) Medium is
Automatically of Desired pH with No User Efforts
[0152] As noted above, all commercially available mammalian cell
culture powdered media require addition of one or more buffer salts
(e.g., sodium bicarbonate) when preparing 1.times. liquid, and then
adjustment of pH, so that the solution will be at proper pH. The
present methods, however, can be used to obviate both the addition
of sodium bicarbonate (as described above in Example 2) and the
need for pH adjustment. In this aspect of the invention, fluid bed
technology is used to introduce acid or base (depending on the
need) to a dry powder medium comprising one or more buffering
salts. In accordance with this aspect of the invention, any
buffering salts or combinations thereof, and any acid or base, may
be used depending upon the desired pH and buffering capacity in the
ultimately reconstituted cell culture medium.
[0153] If sodium bicarbonate is added directly to the DPM as a
powder, it is possible for the end user to simply add water and mix
to yield a solution already containing bicarbonate (see above) and
of proper pH. It is necessary first to determine how much of a pH
adjustment is required. (1) Place 1 L of water in a beaker. Add DPM
to the liquid and mix. (Amount to add/L is given by the
specifications for that powder, e.g., 10 g/L, 13 g/L). In this
case, the weight of the sodium bicarbonate must also be considered
in determining how much to add per liter. (2) After the powder has
dissolved, add 5N HCl to adjust the solution to the desired pH.
Record the amount. (3) Convert this number to amount of 1N HCl.
Calculate how much 1N HCl is needed for adjustment of the total
powder to be agglomerated. (Example: 5 ml of 1N HCl is needed to
adjust 1 L of 1.times. medium A to pH 7.2 from the unadjusted pH of
7.9. That 1 L of 1.times. medium represents, for example, 13.0 g of
DPM. Therefore, for each 13.0 g of DPM, 5 ml of 1N HCl is needed.
If we want to adjust pH of 250 g of DPM, then 250 divided by
13.0=19.2.times.5 ml or 96 ml of 1N HCl is needed to be added to
the powder to make it automatically pH-adjusted.
[0154] This 1N HCl must now be added to the DPM. The best way for
that is to use the injection device, adding 1N HCl instead of
water. In general, the protocol is similar to the above with the
following exceptions: (1) the 1N HCl must be added slowly to the
media which contains sodium bicarbonate. If it is added too
quickly, carbon dioxide may be driven off, resulting in suboptimal
buffering capacity. Because of the volume of 1N HCl generally
required, several 1 minute on, 2 minute off cycles are needed. A
dry powder state must be obtained at the end of each cycle so that
a dynamic system exists where DPM has characteristics of a fluid
process but in reality is a dried powder. (Amazingly, as HCl is
added to the powder, the bulk color changes from dark reddish
purple to light yellow-orange color even though the powder remains
essentially dry at all times due to the continual evaporation
within the system). Since the total amount of HCl has been
calculated to yield an essentially neutral pH, the powder is never
really exposed to "acid" conditions as long as the fluid bed is
properly adjusted (see above; position of the powder particles
within the chamber during operation). It is important to make sure
that all of the powder is moving through the system (i.e., being
lifted, agglomerated and settled continuously) and having no "dead"
zones within the chamber.
[0155] Once the powder is collected after the run, it can be added
to water and reconstituted at any time as long as it has been kept
in proper "dry" packaging and location. No adjustment of pH is
needed. Thus, the invention provides an automatic pH-adjusting dry
powdered medium, where the pH of the liquid medium made by
reconstituting the dry powdered medium requires no adjustment of
pH.
Example 4
Inclusion of Large Molecular Weight Supplements Such as Serum,
Albumin, Hy-Soy, etc, within the DPM Itself
[0156] Heretofore, dried powder media containing serum have not
been commercially available. Using the present methods (via fluid
bed and spray-drying technologies), we have succeeded in adding
serum to a powder in a manner where functionality (cell culture) is
maintained.
[0157] The injection device of the fluid bed apparatus is able to
form a mist with serum, and concentrated albumin. We attempted to
see if serum added to the DPM and dried in this manner would be
functional.
[0158] Procedure for addition of serum: (1) Determine the weight of
standard DPM to be agglomerated. (2) From this, based upon the g/L
for the particular powder, calculate the volume of 1.times. medium
that the g of powder will make. (3) Calculate the volume of serum
that would be needed at a given percentage level of supplementation
(e.g., 100 g of powder to be used in 10 g/L yields 10 L equivalents
of powder). At 5% serum supplementation, 500 ml of serum would be
required to be added by the injection device.
[0159] Protocol for addition of the serum: Serum and albumin are
very viscous. The nozzle spray pattern must be checked for droplet
size and pattern. With the sample tube in the solution to be added
to the powder, test spray against a cardboard or other backdrop.
Check for uniformity and small droplet size. If not a "mist,"
increase atomizing pressure by 0.5 bar and test again. Do this
until sufficient pressure results in a fine mist pattern.
[0160] For use in cell culture applications, it is necessary to
know the weight/ml of serum-DPM to be used per L of 1.times.
medium. To do this, accurately weigh vials or test tubes that will
hold the serum during drying. Place a constant (known) quantity of
serum into each of the vials. Then place vials into a Speed Vac or
lyophilizer. Remove water until dryness. Then weigh the vials
again, this time containing lyophilized serum. Calculate the weight
of serum and express as per ml of original volume. The weight of
agglomerated DPM with serum to use per L will then be the standard
DPM "use" weight plus the weight of the serum at a given level.
[0161] For example, assume that Medium A (DPM) is to be used at 10
g/l. Serum supplementation is to be at 5% v/v. This means that in
addition to the weight of the standard DPM, the weight of the serum
would equal 5%=50 ml to add per L of medium. Assume that serum
powder weighs 0.06 g/ml. Then the weight of the powdered
serum=50.times.0.06 g/L=3 g. Therefore, the weight of
serum-containing DPM that would be added to 1 L of water is the
weight of serum powder (3 g) plus the weight of the standard DPM
(10 g) per liter=13 g/L.
Example 5
Reducing or Eliminating Milling Techniques (High Energy Input
System that Break Components Down to Micron-Sized Particles) When
Manufacturing a DPM
[0162] As noted above, dry powdered medium typically is
manufactured via the milling process, which is laborious and has a
number of problems. The methods of the present invention provide
for the production of a dry powdered medium using fluid bed
technology, which overcomes these labor and technical
constraints.
[0163] A. Blending First in External Device, then Fluid Bed
Treatment
[0164] Normally milled DPM is blended with sodium bicarbonate
(directly as received from the supplier, additional ball milling
not needed). [RPM 1640 with sodium bicarbonate at 2
g/L-equivalents]. This mixture is blended for 20 minutes. The
powder is then placed within the fluid bed chamber and fluidized as
above for bicarbonate-containing media or bicarbonate-containing
media with automatic pH control.
[0165] B. Blending Directly in Fluid Bed Chamber, then
Agglomeration
[0166] Sodium bicarbonate is placed into the chamber directly with
the milled DPM and blended (mixed) for a brief period of time, to
be followed with agglomeration. This eliminates blending in a
separate unit.
[0167] C. Total Elimination of the Ball-Milling Process
[0168] Either all of the DPM chemicals are added directly to the
fluid bed chamber and mixed preliminarily followed by agglomeration
or, more likely, some of the coarser, "stickier", etc. chemicals
are given a brief grinding treatment in a rotary grinder and then
placed within the fluid bed for blending and final
agglomeration.
Example 6
A Method for Having all of the Above Characteristics within this
Same DPM
[0169] We have combined addition of "off the shelf" sodium
bicarbonate with milled DPM and automatic pH control. We have also
combined serum with DPM.
[0170] To combine serum with DPM containing sodium bicarbonate with
automatic pH control, one protocol is to:
[0171] 1. Add sodium bicarbonate (powder, from supplier) to DPM
(milled or ground).
[0172] 2. Blend ingredients (mix, either external unit or fluid
bed).
[0173] 3. In a separate vessel, reconstitute 1 L of the DPM
(containing bicarbonate) with water (1.times.) and determine the
amount of 1N HCl, or 1N NaOH that is required to adjust the pH of
the solution to 7.5. On a liter basis, knowing the mass of powder
to be agglomerated (and thus the L-equivalents), calculate the
amount of 1N HCl or 1N NaOH for the total powder to be agglomerated
at the above-calculated amount. Add this amount via fluid bed
device (injection nozzle). (Although DPM is not "liquid," it is
important to have a powder as close to neutrality as possible but
not of such an acid pH that bicarbonate would be liberated when
adding serum, since moisture is involved in the process. At pH 7.6
or higher, a concentrated solution of sodium bicarbonate will not
evolve CO.sub.2 gas, but at lower pH gas will be given off.)
[0174] 4. Addition of serum (extended agglomeration), based upon
percentage supplementation and g to be agglomerated.
[0175] 5. Using the same 1 L of 1.times. liquid from (3) above,
determine the amount of 1N HCl or 1N NaOH needed to adjust the pH
to the desired pH (e.g., 7.2). Using this information, calculate
the amount to be used for the weight of powder that has been
agglomerated with serum (knowing g/L specifications). Add this
amount via fluid device (injection nozzle).
[0176] 6. Gamma irradiation is used to sterilize the powdered
media.
[0177] In a similar method, a serum-containing DPM may be produced
by combining a particular amount of DPM with a particular amount of
powdered serum (prepared, e.g., by spray-drying as described in
Example 8 below) and then agglomerating the mixture. For example,
for preparation of medium containing 10% powdered FBS, 55.5 g
powdered FBS may be added to 500 g of powdered culture medium and
the powders mixed well by agitation. This mixture may then be
water-agglomerated as described above, and will yield, upon
reconstitution, a culture medium containing 10% FBS which may be
auto-pH-adjusting.
Example 7
Production of 100% Serum Powder by Fluid Bed Processing (to
Simulate Spray-Drying)
Methodology
[0178] 1) We used the benchtop laboratory fluid bed apparatus
(Strea-1). For production of powdered serum, nothing is placed
within the chamber. The lever is used to seal the unit. 2) Serum
was added by way of the injection device (spray unit). As the serum
was added into the chamber, the air flow was increased enough and
the flow of serum slowed enough that evaporation of water occurred
and the serum was dried sufficiently so that powder formed
instantly within the chamber. No moist or fluid coating existed
anywhere within the chamber. 3) Pump speed was set to allow for
.about.1 ml/minute into the chamber. 4) Airflow speed was set to a
setting of .about.8-9. 5) To clean filters intermittently, fan
speed was reduced to .about.2-3. This was done routinely every 5-10
minutes. (The 8-9 airflow setting is so high that the filters will
not blow off the powder and clean themselves). 6) After one round
of filter blow-off, fan speed was increased to previous levels and
the pump turned on. (Once these parameters were set, the pump was
run continuously except when cleaning the filters as indicated). 7)
After all of the serum liquid had been added into the agglomerator,
final drying was performed over five minutes. 8) The filters were
then blown off to collect as much powder as possible, and the
machine shut off and product removed. Powdered serum was placed
into an air-tight container and protected from light.
Typical Instrument Settings
[0179] Drying temperature: 60-65.degree. C. Outlet air temperature:
.about.33.degree. C. Blow out pressure: 5 bar Atomizing pressure:
2.0-2.5 bar Blow back dwell: 2, in between spraying Capacity of
fan: 8-9 throughout run Magnahelics: Filter resistance-150-250,
Resistance of perforated control plate-.about.50, Air volume-less
than 50.
[0180] To determine if agglomeration of the FBS affected the
protein structure or distribution, samples of agglomerated FBS and
liquid FBS were run on SDS-PAGE, stained for protein and scanned
densitometrically. As shown in FIG. 1, agglomerated FBS prepared
according to the present methods (FIG. 1A) demonstrated a nearly
identical protein profile to that observed with liquid FBS (FIG.
1B). These results indicate that the controlled production of dry
powdered FBS by the present methods does not substantially affect
the structure or distribution of the major components of the
serum.
[0181] To determine if agglomeration of the FBS affected its
ability to support cell growth and passage, SP2/0 cells were plated
into DMEM containing either 2% agglomerated ("dry") FBS or 2%
liquid FBS and growth rates and passage recovery examined. As shown
in FIG. 2A, cells plated into media containing agglomerated FBS
demonstrated similar growth kinetics as did cells plated into media
containing liquid FBS. Similarly, cells in media containing
agglomerated FBS recovered from passage with practically identical
growth rates as cells in media containing liquid FBS (FIG. 2B).
Together, these results indicate that the agglomerated FBS of the
present invention performs approximately equivalently to liquid FBS
in supporting growth and passage of cultured cells.
Example 8
Production of 100% Serum Powder by Spray-Drying
[0182] As an alternative to fluid bed processing, the feasibility
of producing dry powdered serum by spray-drying technology was
examined. A three foot diameter laboratory spray drier (Mobile
Minor Spray Drier; NIRO, Columbia, Md.) was used to prepare the
powdered serum. Liquid FBS was aspirated into the spray-dryer and
atomized through a Schlick 940 nozzle located in the middle of the
air dispenser, and the drying air was introduced into the atomizer
through the top air dispenser of the apparatus. Spray drying was
conducted under the following conditions: inlet air
temperature=200.degree. C.; outlet air temperature=70.degree. C.,
atomizing air pressure for the nozzle=2.0 bar; air flow=80.0
kg/hour; spray rate=65 g/minute. During development of these
methods, an initial outlet air temperature of 60.degree. C. was
used; however, this temperature was found to be too low, and the
spray rate was adjusted back to a level to achieve an outlet
temperature of about 70.degree. C. which was found to be optimal.
Following spray-drying, powdered serum was collected at the cyclone
of the apparatus, and process air was filtered through an exhaust
filter prior to recirculation within the apparatus.
[0183] Following production, the powdered serum was characterized
with respect to its physical properties, compared to liquid FBS
from the same source lot. Samples taken from different stages of
the production lot (samples "A" and "B") were reconstituted at a
concentration of 60.44 g/L in endotoxin-free distilled water (Life
Technologies, Inc.), and were examined for endotoxin levels using a
Limulus Amoebocyte Lysate test (Life Technologies, Inc.), for
hemoglobin levels (by spectrophotometrically measuring absorbance
at 525 nm), and by UV/Vis spectrophotometry. Results are shown in
Table 1, and in FIGS. 3A and 3B.
TABLE-US-00001 TABLE 1 Physical Characterization of Powdered Serum.
Material Tested Endotoxin Level (EU/m1) Hemoglobin (mg/100 ml)
Powdered FBS, 0.6 7.7 Sample "A" Powdered FBS, <0.3 7.7 Sample
"B" Liquid FBS <0.3 7.2 (control)
[0184] As seen in Table 1, powdered FBS demonstrated endotoxin and
hemoglobin levels similar to those of the liquid FBS that served as
the source material for production of the powdered FBS. Moreover,
samples taken from different stages of the production process
demonstrated nearly identical endotoxin and hemoglobin levels,
indicating that the present methods result in the production of
material with approximately uniform physical consistency across the
production lot. When samples of powdered and liquid FBS were
examined by UV/visible spectrophotometry (FIG. 3), the trace
observed for powdered FBS (FIG. 3A) was indistinguishable from that
obtained for the source liquid FBS (FIG. 3B). Together, these
results indicate that serum powder prepared by the present
spray-drying methods have nearly identical physical characteristics
as those of liquid sera from which the powders are prepared. Taken
together with those of Example 7 above (see, e.g., FIG. 1), these
results demonstrate that the methods provided by the present
invention result in the production of powdered sera with physical
characteristics that are unaltered from those of the source liquid
sera.
Example 91
Production of Automatically pH-Adjusted Powdered Culture Media
[0185] One reason that sodium bicarbonate is never included in
powdered media is that any moisture, even that in the air, may
result in an acidic condition within the pouch that will result in
the liberation of CO.sub.2 gas. The pouches will become swollen and
produce what have been called "pillows." With fluid bed processing,
the humidity within the apparatus is reduced essentially to
negligible levels prior to the end of the process. We have made
RPMI-1640 powdered media containing sodium bicarbonate and have not
seen evidence of "pillow" formation.
[0186] In order to make a pH-adjusted powdered media, it is
necessary to add the pH-adjusting chemical (usually HCl or NaOH) to
the powder to bring the pH to about 7.0-7.4 upon addition to water.
Once sodium bicarbonate is added to the powder, many powdered media
reconstitute in water on the basic side of neutrality and need HCl
addition. Adding HCl to a powder containing sodium bicarbonate
would be expected to be problematic. However, since the added
liquid (5N HCl in this case) never results in a moistened or
"liquid" state inside the fluid bed apparatus, the sodium
bicarbonate does not give off CO.sub.2 gas and fully retains its
buffering capacity. This has been examined in the present studies
by pH-tittering experiments: equal amounts of acid, in two separate
experiments (FIGS. 4A and 4B) were found to reduce the pH of
agglomerated media and automatic pH-adjusted agglomerated media by
an identical amount as that for a standard medium with sodium
bicarbonate added to the liquid at the time of reconstitution.
These results indicate that both agglomeration with subsequent
adjustment of pH, and agglomeration with adjustment of pH during
the agglomeration process, function equally well to produce
powdered culture media with significant buffering capacity.
Example 10
Effect of Agglomeration on Dissolution Rates of Culture Media
[0187] To examine the effect of agglomeration of culture media on
the rate of dissolution of the media, samples of Opti-MEM I.TM. or
DMEM were agglomerated with water or with FBS (2% only for Opti-MEM
I; 2% or 10% for DMEM). Upon reconstitution of the agglomerated
media in water, the time dissolution of the agglomerated Opti-MEM I
occurred much more quickly than did dissolution of standard
powdered Opti-MEM I (FIG. 5A); results were identical for water-
and FBS-agglomerated Opti-MEM I. Interestingly, however, while
water-agglomerated DMEM dissolved in water much more quickly than
did standard powdered DMEM, the FBS-agglomerated DMEM did not (FIG.
5B).
[0188] Due to the open structure of the agglomerated powdered media
(as opposed to traditional powdered media), capillary action brings
water into close proximity with all of the powder particles. This
prevents the appearance of powder "balls," a complication observed
upon reconstitution of most standard powdered media that leads to
longer dissolution times. In addition to more rapid dissolution,
agglomerated media demonstrated reduced dusting as well. These
results indicate that water-agglomerated culture media, and some
FBS-agglomerated culture media, are much more rapidly dissolving
and generate less dust than traditional powdered culture media.
Example 11
Cell Growth and Subculturing in Reconstituted Agglomerated Culture
Media
[0189] Many uses of culture media require additions of large
molecular weight proteins such as serum or albumin. These molecules
may be in the form of solutions or even powder in the case of
albumin. However, in order to insure uniformity of powdered media,
these proteins are usually added not as a powder but as liquid
after reconstitution of the bulk powdered media to a liquid medium.
This presents some inconvenience since, for example, serum must be
stored in the freezer to maintain performance over time. This adds
expense and inconvenience since the serum must be added aseptically
to the media, increasing chances of contamination. If filtration is
done after addition of serum, another processing step is needed.
There would therefore be advantages to being able to provide serum
as an integral part of the powdered media.
[0190] Therefore, culture media were agglomerated with water or
with various concentrations of FBS. FBS was added to the powdered
media by injecting it into the air-suspended dry powdered media at
high evaporation rates, as generally outlined above. The level of
serum supplementation was 2% in Opti-MEM I media, and 2% or 10% in
DMEM. The growth and passage success of various cell lines in these
media were then assessed.
[0191] As shown in FIG. 6, SP2/0 cells demonstrated similar growth
rates when grown in Opti-MEM I agglomerated with either water or
with FBS (FIG. 6A), compared to cells grown under conventional
culture conditions (liquid serum added to water-reconstituted
powdered media). Similar results were observed with SP2/0 cells
cultured in water- and FBS-agglomerated DMEM supplemented with 2%
FBS (FIG. 6B), and with SP2/0 cells (FIG. 7A), AE-1 cells (FIG. 7B)
and L5.1 cells (FIG. 7C) cultured in water- and FBS-agglomerated
DMEM supplemented with 10% FBS. In addition, SP2/0 cells showed
approximately similar recovery rates from passage when cultured in
water- or agglomerated Opti-MEM I and DMEM supplemented with 2% FBS
(FIGS. 8A and 8B, respectively), as did SP2/0 cells, AE-1 cells and
L5.1 cells cultured in water- and FBS-agglomerated DMEM
supplemented with 10% FBS (FIGS. 9A, 9B and 9C, respectively) and
SP2/0 cells cultured in water-agglomerated DMEM supplemented with
5% FBS (FIG. 10). Furthermore, SP2/0 cells demonstrated identical
passage characteristics in water-agglomerated media produced in
large batches and in automatically pH-adjusting powdered DMEM
containing sodium bicarbonate as they did in standard liquid DMEM
supplemented with 5% FBS (FIG. 10).
[0192] Together, these results indicate that culture media
supplements such as animal sera (e.g., FBS) may be agglomerated
directly into culture media, and that supplementation of culture
media during the agglomeration process in this way produces a
culture medium that provides optimal support of growth and passage
of a variety of cultured cells. Furthermore, these results indicate
that the present culture media powders may be successfully produced
in large batches, including the automatically pH-adjusting media of
the invention that contain sodium bicarbonate.
Example 12
Cell Growth in Culture Media Supplemented with Spray-Dried Serum
Powder
[0193] As a corollary to the experiments shown in Example 7, AE-1
cells and SP2/0 cells were plated into DMEM containing either 2% or
10% spray-dried FBS prepared as described in Example 8, or
containing 2% or 10% liquid FBS, and growth rates and passage
recovery of the cells were examined. Cells were inoculated into
triplicate 25 cm.sup.2 flasks at a density of 1.times.10.sup.5
cells/ml in 10 ml of media. Viable cell density was determined on
days 3-7, and each cell line was tested twice. Results are shown in
FIGS. 11-13.
[0194] As shown in FIG. 11, AE-1 cells cultured in media containing
powdered FBS demonstrated similar growth kinetics to those cells
cultured in media containing standard liquid FBS. As expected, the
cells demonstrated more rapid growth to a higher density in culture
media containing 10% FBS than in media containing 2% FBS, and
demonstrated peak growth by about day four. Similar kinetics were
observed for two separate experiments (FIGS. 11A and 11B),
indicating that these results were reproducible. Analogous results
were obtained in two experiments in which the growth rates of SP2/0
cells were measured in media containing powdered or liquid FBS
(FIGS. 12A and 12B). In addition, AE-1 cells cultured in media
containing 5% powdered FBS recovered from passage with identical
growth rates as cells in media containing liquid FBS (FIG. 13).
[0195] These results indicate that the powdered FBS prepared by the
spray-drying methods of the present invention performs
approximately equivalently to liquid FBS in supporting growth and
passage of cultured cells. Together with those from Examples 7 and
8, these results indicate that the methods of the present invention
may be used to produce powdered FBS, by fluid bed or spray-drying
technologies, that demonstrates nearly identical physical and
performance characteristics as those of liquid FBS.
Example 13
Effect of Irradiation on Performance of Agglomerated Media
[0196] Recently, concerns have been raised about the biological
purity of media and media components (including supplements) used
for bioproduction, particularly in the biotechnology industry.
Gamma irradiation is a sterilization process that is known to work
well with certain liquids and powders that are not typically
amenable to sterilization by heat or toxic gas exposure. Therefore,
samples of water- or FBS-agglomerated culture media were .gamma.
irradiated with a cobalt source at 25 kGy for up to several days,
and the growth rates of various cell types examined.
[0197] In one set of experiments, SP2/0 cells were inoculated into
various media at 1.times. cells/ml and cultured at 37.degree. C. At
various intervals, samples were obtained aseptically and cell
counts determined by Coulter counting and viability determined by
trypan blue exclusion. Media were prepared by dissolving sufficient
powdered media to make a 1.times. solution in 1 L of water,
stirring and filtering through a 0.22 .mu.m filter. Results are
shown in the graph in FIG. 14. Those conditions on the graph that
state "pwdr FBS" on the graph refer to the addition of powdered FBS
(prepared as in Examples 7 or 8 above) to the reconstituted
1.times. medium prepared from either standard powdered media or
from agglomerated media (irradiated or non-irradiated). Those
conditions on the graph that state "Irradia. agglom. DMEM+FBS"
refer to use of the fluid bed to make the agglomerated media by
spraying FBS into the powdered media (standard or agglomerated) to
make an FBS-agglomerated media.
[0198] As shown in FIG. 14, .gamma. irradiation of standard
powdered basal media and agglomerated basal media did not
deleteriously affect the ability of these media to support SP2/0
cell growth. In addition, while irradiation did negatively impact
powdered media containing powdered FBS, and powdered FBS itself,
this effect diminished with increasing serum concentration.
[0199] To more broadly examine these .gamma. irradiation effects,
samples of VERO cells were inoculated into VP-SFM.TM. that had been
conventionally reconstituted or agglomerated as above. To the
powdered media in the agglomeration chamber, however, epidermal
growth factor (EGF) and ferric citrate chelate, traditional
supplements for this media, were added via the spray nozzle during
agglomeration. Media were then used directly or were .gamma.
irradiated as described above. Cells were inoculated at
3.times.10.sup.5 cells/flask into T-25 flasks and incubated at
37.degree. C. Cell counts and viability were performed as described
above, with results shown in FIG. 15.
[0200] As seen in FIG. 15, VERO cells demonstrated approximately
equivalent growth and passage success when cultured in agglomerated
media that had been .gamma.-irradiated as in agglomerated media
that had not been .gamma.-irradiated. Furthermore, irradiation of
the media had no effect on the low-level culture supplements EGF
and ferric citrate chelate that were present in the media.
[0201] These results indicate that .gamma. irradiation may be used
as a sterilization technique in the preparation of many bulk
agglomerated culture media, including those containing serum, EGF
or other supplements, by the present methods.
Example 14
Effect of Irradiation on Performance of Powdered Media
Supplements
[0202] To demonstrate the efficacy of the present methods in
producing sterile media supplements, lyophilized human
holo-transferrin was irradiated by exposure to a cobalt .gamma.
source at 25 kGy for about 3 days at -70.degree. C. or at room
temperature 293 cells were then cultured in media that were
supplemented with irradiated transferrin or with control
transferrin that had not been irradiated (stored at -70.degree. C.
or at room temperature), and cell growth compared to that of
standard transferrin-containing culture media or media that
contained no transferrin.
[0203] Mid-log phase 293 cells that were growing in serum-free 293
medium (293 SFM) were harvested, washed once at 200.times.g for 5
minutes and resuspended in transferrin-free 293 SFM for counting
and viability determination. Cells were plated into triplicate 125
ml Ehrlenmeyer flasks at a density of 3.times.10.sup.5 cells/ml in
a volume of 20 ml in 293 SFM (positive control), transferrin-free
293 SFM (negative control), in 293 SFM containing non-irradiated
transferrin stored at -70.degree. C. or at room temperature, or in
293 SFM containing irradiated transferrin prepared as described
above. Flasks were placed into a rotary shaker set at about 125
rpm, in a 37.degree. C. incubator equilibrated with an atmosphere
of 8% CO.sub.2/92% air. Daily cell counts were determined using a
Coulter particle counter and viabilities were determined by trypan
blue exclusion according to standard procedures. When the cells
reached a density of about 1.2 to 1.7.times.10.sup.6 per flask, the
contents of one of the flasks of each sample were harvested,
centrifuged, resuspended into fresh medium and passaged into three
new flasks. Cell counts and viabilities of the previous and next
passages were then performed as described above. Four consecutive
passages of cells incubated under the above conditions were
tested.
[0204] As shown in FIGS. 16A-16D, cells cultured in media
containing transferrin that was .gamma. irradiated at either
-70.degree. C. or at room temperature demonstrated nearly identical
growth kinetics and survival in the first passage (FIG. 16A),
second passage (FIG. 16B), third passage (FIG. 16C) and fourth
passage (FIG. 16D) as did cells cultured in standard 293 SFM or in
293 SFM containing transferrin that had not been .gamma.
irradiated. Cells cultured in transferrin-free media, however,
survived well during the first passage (FIG. 16A) but stopped
growing and demonstrated a significant loss in viability upon
subculturing (FIG. 16B).
[0205] These results demonstrate that .gamma. irradiation may be
used as a sterilization technique in the preparation of bulk
powdered culture media supplements, such as transferrin, in the
methods of the present invention. Furthermore, these data indicate
that culture media supplements such as transferrin may be .gamma.
irradiated at room temperature without significant loss of
activity.
Example 15
Effect of Irradiation on Biochemical Characteristics of Powdered
Sera
[0206] To further determine the impact of .gamma. irradiation on
sera, samples of spray-dried powder FBS were irradiated at 25 kGy
at -70.degree. C. or at room temperature (RT), and were analyzed
commercially for the concentrations of various biochemical
constituents in the sera. As controls, samples of non-irradiated
spray-dried FBS and liquid FBS were also analyzed. Results are
shown in Table 2.
TABLE-US-00002 TABLE 2 Chemical Analysis of Spray-Dried FBS Dried
FBS, Dried Irr. @ FBS, Irr. Non-irradiated Liquid Reference
Constituent -70.degree. C. @RT Dried FBS FBS Units Range Sodium 139
137 139 140 mM 136-144 Potassium 13.2 13.2 13.0 13.2 mM 3.6-5.2
Chloride 98 97 98 100 mM 98-108 Uric Acid 1.6 1.3 1.7 1.9 mg/dL
2.2-8.3 Phosphorus 10.1 10.1 9.6 10.2 mg/dL 2.2-4.6 Calcium 14.9
14.8 14.8 14.5 mg/dL 8.6-10.2 Ionizable >5.5 >5.5 >5.5
>5.5 mg/dL 3.8-4.5 Calcium Magnesium 2.77 2.76 2.75 2.76 meg/L
1.4-2.0 Alkaline 57 47 68 269 U/L 31-142 Phosphatase Gamma GT 3 5
<5 5 U/L 1-60 (GGTP) AST (SGOT) 7 5 5 33 U/L 1-47 ALT (SGPT) 5
<5 <5 7 U/L 1-54 LD 56 <50 50 510 U/L 110-250 Total
Bilirubin 0.19 0.24 0.22 0.13 mg/dL 0.2-1.4 Direct 0.04 0.07 0.07
0.04 mg/dL 0.0-0.3 Bilirubin Glucose 67 38 39 88 mg/dL 65-125 BUN
15 15 15 15 mg/dL 6-23 Creatinine 2.98 3.08 3.1 2.77 mg/dL 0.1-1.7
BUN/Creatine 5.0 4.9 4.8 5.4 -- 7.0-20.0 Ratio Total Protein 3.6
3.6 3.5 3.7 gm/dL 6.4-8.1 Albumin 2.7 2.7 2.8 2.8 gm/dL 3.7-5.1
Globulin 0.9 0.9 0.7 0.9 gm/dL 2.1-3.6 Albumin/Globulin 3.0 3.0 4.0
3.1 -- 1.1-2.3 Ratio Cholesterol 30 30 32 30 mg/dL <200 HDL 28
30 30 27 mg/dL 39-90 Cholesterol Chol/HDL 1.07 1.00 1.07 1.11 --
<4.5 Ratio Triglycerides 72 74 72 73 mg/dL 30-200 Iron 213 217
214 186 meg/dL 40-175 Plasma Hb 13.3 11.5 13.7 22.6 mg/dL
3.4-20.5
[0207] These results indicate that the .gamma. irradiation process
did not significantly affect the concentrations of most of the
biochemical constituents of FBS. These results also indicate that
upon spray-drying, several of the components of FBS (alkaline
phosphatase, AST, and LD, and possibly glucose) undergo a
significant reduction in concentration compared to their
concentrations in the starting liquid FBS.
Example 16
Effects of Irradiation on Performance of Powdered Sera
[0208] To examine the impact of .gamma. irradiation on the ability
of dried powder sera to support cell growth, samples of spray-dried
FBS irradiated under various conditions were used to supplement
culture media, and adherent and suspension cells were grown for up
to three passages in these media. As model suspension cells, the
hybridoma lines SP2/0 and AE-1 were used, while VERO and BHK
cultures were used as typical adherent cells. Cells were cultured
in media containing test sera or control sera (spray-dried but not
irradiated) for up to three passages according to the general
procedures outlined in Example 14 above. At each passage point,
cells were harvested and subcultured, while an aliquot was counted
as above for viable cells/ml. Results at each point were expressed
as a percentage of the viable cell count obtained in media
supplemented with liquid FBS, and are shown in FIGS. 17A, 17B, 17C
and 17D.
[0209] Several conclusions may be drawn from the results of these
studies. First, .gamma. irradiation of FBS does not appear to
reduce the ability of spray-dried FBS to support the growth of
suspension and adherent cells (compare the irradiated data sets to
the non-irradiated data set in each figure). In fact, BHK cells
(FIG. 17D) actually grew better in media containing powdered FBS
that had been irradiated at -70.degree. C. than they did in
non-irradiated sera. Second, sera irradiated at -70.degree. C.
appear to perform better than those irradiated at room temperature
in their ability to support cell growth, except perhaps for VERO
cells (FIG. 17C). Finally, the results of these studies were very
cell type-specific: suspension cells (FIGS. 17A and 17B) grew
better in spray-dried FBS, irradiated and non-irradiated, than did
adherent cells (FIGS. 17C and 17D); and among adherent cells, BHK
cells (FIG. 17D) grew better in spray-dried FBS than did VERO cells
(FIG. 17C).
[0210] These results demonstrate that .gamma. irradiation may be
used as a sterilization technique in the preparation of bulk
powdered sera, such as FBS, in the methods of the present
invention. Furthermore, unlike those reported for transferrin in
Example 14 above, these data suggest that the optimal temperature
for irradiation of sera, in order to maintain the ability of the
sera to support cell growth, is likely to be below room
temperature.
[0211] Having now fully described the present invention in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious to one of ordinary skill in
the art that the same can be performed by modifying or changing the
invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof, and that such
modifications or changes are encompassed within the scope of the
appended claims.
[0212] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
* * * * *