U.S. patent application number 13/092016 was filed with the patent office on 2011-10-27 for cell culture medium comprising small peptides.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Shawn BARRETT, Scott JACOBIA.
Application Number | 20110262965 13/092016 |
Document ID | / |
Family ID | 44816118 |
Filed Date | 2011-10-27 |
United States Patent
Application |
20110262965 |
Kind Code |
A1 |
BARRETT; Shawn ; et
al. |
October 27, 2011 |
CELL CULTURE MEDIUM COMPRISING SMALL PEPTIDES
Abstract
Cell culture media, concentrated media and feeds, methods of
manufacturing cell culture media and feeds, and methods of
culturing cells are provided. One or more small peptides, including
dipeptides are added to the cell culture media to provide improved
stability and improved conditions for culturing cells.
Inventors: |
BARRETT; Shawn; (Clarence
Center, NY) ; JACOBIA; Scott; (Snyder, NY) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
44816118 |
Appl. No.: |
13/092016 |
Filed: |
April 21, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61327644 |
Apr 23, 2010 |
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Current U.S.
Class: |
435/69.1 ;
435/235.1; 435/253.6; 435/255.7; 435/404; 435/431 |
Current CPC
Class: |
C12N 5/005 20130101;
C12N 5/0018 20130101; C12N 2510/02 20130101; C12N 2500/32 20130101;
C12P 21/02 20130101; C12N 2501/998 20130101 |
Class at
Publication: |
435/69.1 ;
435/404; 435/431; 435/255.7; 435/253.6; 435/235.1 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C12N 7/00 20060101 C12N007/00; C12N 1/20 20060101
C12N001/20; C12N 5/02 20060101 C12N005/02; C12N 1/16 20060101
C12N001/16 |
Claims
1. A cell culture medium comprising at least one small peptide,
said peptide comprising at least two amino acids, wherein at least
one of the amino acids is a cysteine or a tyrosine.
2. The cell culture medium of claim 1, wherein at least one of the
remaining amino acids of the small peptide are selected from the
group consisting of alanine, glycine, serine, valine, proline,
aspartic acid, and glutamic acid.
3. The cell culture medium of claim 2, wherein at least one of the
remaining amino acids of the small peptide is alanine or
glycine.
4. The cell culture medium of claim 1, wherein at least one of the
small peptides is a dipeptide selected from the group consisting of
X-tyrosine, X-cysteine, tyrosine-X, and cysteine-X, or a salt
thereof, and wherein X is selected from the group consisting of
alanine, glycine, serine, valine, proline, aspartic acid, and
glutamic acid, and/or, and wherein the N-terminal amino acid of the
one or small peptides has a free amino group.
5. The cell culture medium of claim 1, wherein X is alanine or
glycine.
6. The cell culture medium of claim 1, wherein the cell culture
medium is a liquid solution.
7. The cell culture medium of claim 1, wherein the cell culture
medium is a dry powder or a granulated dry powder.
8. The cell culture medium of claim 1, further comprising a
carbohydrate and an amino acid, or a salt thereof.
9. The cell culture medium of claim 8, wherein the carbohydrate is
a hexose.
10. The cell culture medium of claim 8, wherein the amino acid, or
salt thereof, is one or more of arginine, asparagine, aspartic
acid, cysteine, glutamic acid, histidine, isoleucine, lysine,
methionine, phenylalanine, proline, hydroxyproline, serine,
threonine, tryptophan, tyrosine, and valine.
11. The cell culture medium of claim 1, further comprising a
vitamin, a salt, a buffering agent, or an inorganic element.
12. The cell culture medium of claim 1, wherein the cell culture
medium does not contain a lipid, a hydrolysate or a fraction
thereof, or a growth factor.
13. The cell culture medium of claim 4, wherein the cell culture
medium does not contain a protein.
14. The cell culture medium of claim 4, wherein the cell culture
medium is concentrated as a 2.times. or greater formulation.
15. The cell culture medium of claim 4, wherein the one or more
dipeptides are alanyl tyrosine, and/or alanyl cysteine, and/or
alanyl cystine dimer.
16. The cell culture medium of claim 4, wherein the one or more
dipeptides are present at a concentration of about 1 g/L to about
16 g/L.
17. The cell culture medium of claim 4, wherein the one or more
dipeptides are present at a concentration of about 2.5 g/L to about
8.5 g/L.
18. The cell culture medium of claim 6, wherein the liquid solution
stored at 2-8.degree. C. remains free of precipitate for over 12
months.
19-49. (canceled)
50. The cell culture medium of claim 9, wherein the hexose is
selected from the group consisting of glucose, galactose, fructose
and maltose.
51. A cell culture medium, concentrated feed, or reconstituted
medium comprising a small peptide comprising cysteine or tyrosine,
wherein said cell culture medium, concentrated feed, or
reconstituted medium is serum-free, protein-free and/or
hydrolysate-free.
52. The cell culture medium, concentrated feed, or reconstituted
medium of claim 51 wherein said small peptide comprises two, three,
four, five or six amino acids.
53. The cell culture medium, concentrated feed, or reconstituted
medium of claim 1 that supports the production of a recombinant
protein.
54. The cell culture medium, concentrated feed, or reconstituted
medium of claim 53 wherein the recombinant protein is an
immunoglobulin or a fragment thereof.
55. The composition comprising the cell culture medium,
concentrated feed, or reconstituted medium of claim 1 and a
cell.
56-62. (canceled)
63. Use of the cell culture medium, concentrated feed, or
reconstituted medium of claim 1 to produce a virus or a recombinant
protein.
64. (canceled)
65. The cell culture medium of claim 1, wherein the small peptide
is either a dipeptide, a tripeptide, a tetrapeptide, a
pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a
nonapeptide, or a decapeptide.
66. The cell culture medium of claim 1, wherein the small peptide
is at least about two to ten amino acids in length.
67. The cell culture medium of claim 1, wherein the small peptide
is at least about ten amino acids in length.
68. (canceled)
Description
BACKGROUND
[0001] Cell culture media provide the nutrients necessary to
maintain and grow cells in a controlled, artificial and in vitro
environment. Nutrient formulations, pH, and osmolality of cell
culture media vary in accordance with parameters such as cell type,
cell density, and the culture system employed.
[0002] Media formulations have been used to cultivate a number of
cell types including animal, plant and bacterial cells. Cultivated
cells have many uses including the study of physiological processes
and the production of useful biological substances. Examples of
such useful products include polypeptides, such as monoclonal
antibodies, hormones, growth factors, enzymes, and other
polypeptides of interest. Such products have many commercial and
therapeutic applications and, with the advent of recombinant DNA
technology, cells can be engineered to produce large quantities of
these products. Cultured cells are also routinely used for the
isolation, identification and growth of viruses which can be used
as vectors and/or vaccines. 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.
[0003] 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 (1980);
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,
minerals, 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.
[0004] Glutamine is routinely used in cell culture media because it
has been shown to be a major energy source for cultured cells. In
1959, Eagle showed that the amount of glutamine necessary for
optimal growth of mammalian cell cultures is 3 to 10 times greater
than the amount of other amino acids. Eagle et al., Science
130:432-37 (1959). However, glutamine is unstable in aqueous
solution and at elevated temperatures, forming pyroglutamate and
ammonia, which can be toxic to certain cells. Roth et al., In Vitro
Cellular & Developmental Biology 24(7):696-98 (1988).
Therefore, glutamine is typically added to cell culture medium
immediately before use.
[0005] Alternatively, to avoid the formation of toxic substances
like pyroglutamate and ammonia, glutamine-containing dipeptides,
such as alanyl-glutamine or glycyl-glutamine, can be used in cell
culture media instead of glutamine Roth et al., In Vitro Cellular
& Developmental Biology 24(7):696-98 (1988). Glutamate has also
been used instead of glutamine to reduce the accumulation of
ammonia in the cell culture medium See Cell Culture Technology for
Pharmaceutical and Cell-Based Therapies, 52, Sadettin Ozturk and
Wei-Shou Hu eds., Taylor and Francis Group (2006).
[0006] Others have proposed acylating a dipeptide, such as alanyl
glutamine, to make the dipeptide more stable under heat
sterilization conditions. For example, U.S. Pat. No. 5,534,538
describes N-acyl dipeptides for use in enteral or parenteral
nutrition where the N-acyl dipeptides are more stable under heat
sterilization conditions than corresponding non-acylated dipeptides
and where the N-acyl group advantageously delays splitting of the
dipeptide until it reaches the kidneys. U.S. Pat. No. 5,534,538
also found that N-acyl alanyl glutamine was a suitable glutamine
source for cell culture media, similar to the corresponding
non-acylated dipeptide (alanyl glutamine), which was a known
component of cell culture medium (see Roth et al. above). U.S. Pat.
No. 5,534,538 further described the N-acyl dipeptides as
advantageous over free dipeptides, at least in part, because of the
increased stability of N-acyl dipetides under heat sterilization
conditions. Thus, U.S. Pat. No. 5,534,538 actually teaches away
from using dipeptides where the N-terminal amino acid has a free
amino group.
[0007] Applicants have found that certain amino acids, like
tyrosine, have limited solubility at the desired concentration for
maximal cell growth or protein production. Applicants have also
found that other amino acids, like cysteine, are unstable and prone
to precipitation over time in aqueous cell culture medium,
particularly in concentrated cell culture medium. Specifically,
because cysteine has a thiol group, it is susceptible to an
oxidation reaction in which two cysteine residues are linked
together by a disulfide bond to form cystine
(SCH.sub.2CH(NH.sub.2)CO.sub.2H).sub.2. Cystine has low solubility
in water and readily precipitates out of solution. As a result, it
has not been possible to produce a shelf-stable, liquid cell
culture medium containing concentrations of tyrosine and cysteine
required for maximal cell growth and/or protein production.
[0008] Applicants have also tried to address this problem by using
less than the desired concentration of tyrosine or cysteine in the
aqueous cell culture medium. In so doing, it is possible to achieve
an acceptable aqueous shelf life. However, this is accomplished at
the expense of optimal cell growth, protein production or viral
production. In other words, cell culture medium with lower
concentrations of tyrosine and cysteine supports reduced cell
growth and/or protein production as compared to cell culture medium
with the optimal concentrations of tyrosine and cysteine.
[0009] Thus, there exists a current need for a medium, concentrated
medium or concentrated feed supplement containing amounts of
tyrosine and cysteine sufficient to support maximal cell growth
and/or protein or viral production while avoiding the problems
caused by the limited solubility of tyrosine or the limited
stability of cysteine, including the tendency of cysteine to
precipitate out of solution over time. In addition, there exists a
need for such a feed supplement to be in a concentrated form, so
that its addition would not greatly add volume to the final cell
culture system. There also exists a need that when such nutrient
feeds are added, the pH and osmolality of the resulting system be
automatically balanced, and that the medium or concentrated feed be
available in a liquid or a dry format. Lastly, there exists a need
for a single-regimen, concentrated feed supplement comprising
amounts of cysteine and tyrosine sufficient to support maximal cell
growth and/or protein production, and/or expressed protein of high
quality.
SUMMARY
[0010] The compositions of the invention are directed, in part, to
cell culture media, concentrated media, or concentrated feed
supplements comprising concentrations of cysteine and tyrosine that
support maximal cell growth and/or protein, or viral production
while avoiding the problems caused by their limited solubility and
stability. In one aspect, the media, concentrated feed supplements,
or concentrated media may be serum-free compositions. In one
aspect, the compositions may comprise human serum components, like
human serum albumin. In a further aspect, the human serum albumin
may be recombinant, (r-human serum albumin), derived from
recombinant sources, in some preferred cases, form plant sources
like rice, corn, wheat, potato, or from fungal sources, or from
yeast or other equivalent microorganisms well-known to be used in
the art (xeno-free culture). In another aspect, the media,
concentrated feed supplements, or concentrated media may be
protein-free compositions. In yet another aspect, the media,
concentrated feed supplements, or concentrated media may be
protein-hydrolysate free compositions, and further, may be free of
fractions of protein-hydrolysates. In a certain aspect, the media,
concentrated feed supplements, or concentrated media may be
serum-free, protein-free, and protein hydrolysate-free
compositions. In a preferred aspect, these compositions may
comprise components that are chemically-defined, serum-free,
protein-free and free of any protein hydrolysates, or fractions
thereof. In certain embodiments, the cell culture medium,
concentrated medium or cell culture feed supplement having one or
more small peptides, or one or more dipeptides, further does not
contain one or more of the following: lipids, hydrolysates or a
fraction thereof, or growth factors.
[0011] The invention is also directed, in part, to methods for
analyzing the compositions described above for the presence or
absence of a short peptide comprising cysteine or tyrosine. The
analysis of the media is performed by any known method in the art,
for instance, by mass spectrometry (LCMS), capillary
electrophoresis or HPLC.
[0012] In particular, the present disclosure is directed to a cell
culture medium, a concentrated feed, or a cell culture supplement
comprising one or more small peptides having two to six amino
acids, as defined later in the application. Therefore, the present
disclosure provides a cell culture medium, a concentrated feed, or
a cell culture supplement, wherein the medium, feed or supplement
comprises one or more small peptides selected from
X.sub.1-5-tyrosine, X.sub.1-5-cysteine, tyrosine-X.sub.1-5, and
cysteine-X.sub.1-5, or any small peptide where cysteine or tyrosine
is anywhere within the short peptide of 1 to 6 amino acids (for
e.g., X-cysteine-X.sub.1-4, X-tyrosine-X.sub.1-4, etc.) or a salt
thereof, wherein X is any amino acid, and wherein the N-terminal
amino acid of the short peptide has a free amino group. In one
embodiment, X is alanine or glycine. In another embodiment X is
serine, valine, proline, aspartic acid, or glutamic acid. In a
particular embodiment, the present disclosure provides a cell
culture medium, a concentrated feed, or a cell culture supplement,
wherein the medium, feed or supplement comprises one or more
dipeptides selected from X-tyrosine, X-cysteine, tyrosine-X, and
cysteine-X, or a salt thereof, wherein X is any amino acid, and
wherein the N-terminal amino acid of the dipeptide has a free amino
group. In one embodiment, X is alanine or glycine. In another
embodiment X is serine, valine, proline, aspartic acid, or glutamic
acid. In yet another embodiment, the one or more dipeptides are
alanyl tyrosine and/or alanyl cysteine. The cell culture medium,
concentrated feed, or cell culture supplement may be a liquid
solution or a dry powder, such as a dry powder media (DPM) or an
agglomerated powder (AGT.TM.). In one embodiment, the liquid
solution is stored at 2-8.degree. C. and remains free of
precipitate for over 12 months.
[0013] In one embodiment of the invention, the concentration of
tyrosine (as comprised within the small peptide), in solution, in a
medium, feed or supplement, will be greater than the concentration
of tyrosine that would remain soluble in an identical solution, if
the tyrosine were present as a monomer (that is, tyrosine in a
small peptide may make the solution "supersaturated for tyrosine").
In one embodiment, the solution will have between about 1 and at
least about 100 times, or about 1 and at least about 25 times, the
soluble tyrosine concentration as would be capable with the
monomer. For example, the concentration of small peptides or
dipeptides in some cell culture media, concentrated feeds or
supplements may be about 1 to at least about 5 times, about 1 to at
least about 10 times, about 1 to at least about 15 times, about 1
to at least about 20 times, about 1 to at least about 30 times,
about 1 to at least about 40 times, about 1 to at least about 50
times, about 1 to at least about 60 times, about 1 to at least
about 70 times, about 1 to at least about 80 times, about 1 to at
least about 90 times, about 1 to at least about 100 times, about 10
to at least about 20 times, about 10 to at least about 30 times,
about 10 to at least about 40 times, about 10 to at least about 50
times, about 10 to at least about 60 times, about 10 to at least
about 70 times, and so on, the soluble tyrosine concentration as
would be capable with the monomer.
[0014] In one embodiment of the invention, the concentration of
cysteine (as comprised within the small peptide), in solution, in a
medium, feed or supplement, will be greater in the solution than
the concentration of cysteine that would remain soluble in an
identical solution if the cysteine were present as a monomer (that
is, cysteine in a small peptide may make the solution
"supersaturated for cysteine"). In one embodiment, the solubility
of cysteine (or lack thereof) would include, for example, loss of
solubility caused by conversion of cysteine to cystine. In one
embodiment, the solution will comprise at least, between about 1
and about 25 times, the soluble cysteine concentration capable with
the monomer. For example, the concentration of small peptides or
dipeptides in some cell culture media, concentrated feeds or
supplements may be at about 1 to at least about 5 times, about 1 to
at least about 10 times, about 1 to at least about 15 times, about
1 to at least about 20 times, about 1 to at least about 30 times,
about 1 to at least about 40 times, about 1 to at least about 50
times, about 1 to at least about 60 times, about 1 to at least
about 70 times, about 1 to at least about 80 times, about 1 to at
least about 90 times, about 1 to at least about 100 times, about 1
to at least about 110 times, about 10 to at least about 20 times,
about 10 to at least about 30 times, about 10 to at least about 40
times, about 10 to at least about 50 times, about 10 to at least
about 60 times, about 10 to at least about 70 times, about 10 to at
least about 80 times, about 10 to at least about 90 times, about 10
to at least about 100 times, about 10 to at least about 110 times,
and so on, the soluble cysteine concentration as would be capable
with the monomer. In one embodiment, the solution is supersaturated
for both tyrosine and cysteine.
[0015] In one embodiment, the solution will be supersaturated for
both tyrosine and cysteine in, for example, the ranges described
above for each of tyrosine or cysteine alone.
[0016] In one embodiment, the addition of the concentrated feeds,
media or supplements described above could reduce the volume of
supplementation to a culture system, for e.g., a fed-batch culture,
which may already be in progress. The culture may be in progress
for a few hours to a few days. In another embodiment, increasing
the solubility of the cysteine and tyrosine comprised within small
peptides allows for the design of single part concentrated feeds,
which may also be pH neutral, which is part of the invention. The
invention is directed in part to preparation of single regimen
concentrated feeds, concentrated media and concentrated supplements
comprising cysteine and tyrosine comprised within a small peptide,
to increase their solubility and stability within the
composition.
[0017] The cell culture medium, concentrated feed or cell culture
supplement having one or more small peptides, or one or more
dipeptides optionally comprises one or more of the following: a
carbohydrate, a vitamin, a salt, an inorganic element, a buffering
agent, and an amino acid, or a salt thereof. In one embodiment the
carbohydrate is a hexose sugar. Alternately, pentose, hexose, or
their derivatives or other equivalents may be used. In some
instances, oligosaccharides or their derivatives may be used; or,
the hexose may be selected from the group consisting of glucose,
galactose, fructose and maltose. In a particular embodiment, the
carbohydrate is glucose.
[0018] In one embodiment, the amino acid, or salt thereof, is one
or more of arginine, asparagine, aspartic acid, cysteine, glutamic
acid, glutamine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, proline, serine, threonine, tryptophan,
tyrosine, and valine.
[0019] In one embodiment, the cell culture medium, concentrated
feed or cell culture supplement comprises a 1) first dipeptide,
X-tyrosine, or a salt thereof, and a second dipeptide, X-cysteine,
or a salt thereof, wherein X is alanine or glycine and wherein the
alanine or glycine has a free amino group, 2) a carbohydrate, such
as glucose, and 3) an amino acid, or a salt thereof. The amino
acid, or a salt thereof, may comprise one or more of arginine,
asparagine, aspartic acid, cysteine, glutamic acid, histidine,
isoleucine, leucine, lysine, methionine, phenylalanine, proline,
serine, threonine, tryptophan, tyrosine, and valine.
[0020] In one embodiment, the cell culture medium, concentrated
feed or cell culture supplement has one or more small peptides, or
one or more dipeptides, suitable for culturing a bacterial cell, a
yeast cell, a plant cell, or an animal cell, such as an insect cell
(e.g., Drosophila cells, Spodoptera cells or Trichoplusia cells), a
nematode cell (e.g., C. elegans cells) or a mammalian cell (e.g.,
CHO cells, COS cells, VERO cells, BHK cells, AE-1 cells, SP2/0
cells, L5.1 cells, PerC6, hybridoma cells, HEK 293, including human
cells).
[0021] In certain embodiments, the cell culture medium having one
or more small peptides or one or more dipeptides is a 1.times.
formulation. In other embodiment, the cell culture medium is
concentrated as a 2.times. or greater formulation, as discussed
elsewhere in this application. In certain embodiments, the one or
more small peptides or dipeptides, such as X-tyrosine, X-cysteine,
tyrosine-X, or cysteine-X, are present in the cell culture medium
at a concentration of about 1 g/L to about 16 g/L, about 1 g/L to
about 10 g/L, about 1 g/L to about 5 g/L, about 2.5 g/L to about 16
g/L, about 2.5 g/L to about 10 g/L, about 2.5 g/L to about 5 g/L,
or about 2.5 g/L to about 8.5 g/L.
[0022] In another aspect, the disclosure provides a method of
culturing a cell, comprising contacting the cell with the small
peptide- or dipeptide-containing cell culture medium described
herein under conditions supporting 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. In one
embodiment, the animal cell for culturing according to the present
methods is an insect cell (e.g., Drosophila cells, Spodoptera cells
or Trichoplusia cells), a nematode cell (e.g., C. elegans cells) or
a mammalian cell (e.g., CHO cells, COS cells, VERO cells, BHK
cells, AE-1 cells, SP2/0 cells, L5.1 cells, PerC6, hybridoma cells,
HEK 293 or other human cells).
[0023] In another embodiment, the method of culturing a cell
comprises contacting the cell with a basal cell culture medium
under conditions supporting the cultivation of the cell and
supplementing the basal cell culture medium with a cell culture
medium, a cell culture supplement, or a cell culture feed, having
one or more small peptides or one or more dipeptides, as described
herein. In one embodiment, the one or more dipeptides selected from
X-tyrosine, X-cysteine, tyrosine-X, or cysteine-X, or a salt
thereof, wherein X is selected from alanine, glycine, serine,
valine, proline, aspartic acid, or glutamic acid and wherein the
N-terminal amino acid of the one or dipeptides has a free amino
group. In another embodiment, the one or more dipeptides are alanyl
tyrosine and/or alanyl cysteine.
[0024] In one embodiment, the basal cell culture medium is
supplemented with a solution, comprising cysteine and tyrosine in a
small peptide, on more than one day, wherein the solution may be a
concentrated medium, or a concentrated feed supplement. In certain
embodiments, applicants may refer to the use of media, feed,
supplements, or other solutions which comprise small peptides
(including, for example, solutions comprising dipeptides which
comprise at least one tyrosine or cysteine residue). Indeed, one
embodiment of applicants' invention is the use of such solutions in
the culturing of cells. In that respect, one of skill in the art
will appreciate that in certain recitations herein, Applicants may
refer to a specific supplement, media(um), feed or solution merely
as an example of an embodiment that could equally apply to any of
the types of solutions described herein. For example, one of skill
in the art would understand that a recitation of a "supplement"
with a specific amino acid composition can equally describe a
"feed" with a like composition and vice versa.
[0025] The basal cell culture medium, concentrated medium,
concentrated feed or supplement may comprise a small peptide- or
dipeptide-containing medium or feed on day 0, day 1, day 2, day 3,
day 4 or 5 days after starting the cell culture and is supplemented
daily thereafter through day 13 or 14 or until the viability of the
culture drops below a pre-determined level (eg. 50%). The basal
cell culture medium is optionally supplemented with the small
peptide- or dipeptide-containing cell culture medium at about 2% of
the total volume of the basal cell culture medium. In one
embodiment, the cell produces a protein, a peptide, a small RNA
(for e.g., miRNA, siRNA, etc). By protein is meant, a recombinant
protein or a naturally occurring protein. By protein, whether
recombinant or natural, is also meant a full-length protein or a
part thereof (like a domain, a motif, a polypeptide chain or
polypeptide fragment). By protein, whether recombinant or natural,
is also meant an intracellular protein, an extracellular protein, a
secreted protein, hormone, cytokine, receptor, extracellular matrix
protein, an immunoglobulin or a part of the immunoglobulin, or a
fragment thereof. Preferably, the protein yield may be higher with
the cell culture medium, feed or supplement comprising the short
peptides, or dipeptides of the invention. In a specific aspect, for
instance, the protein yield for an immunoglobulin may be greater
than 3000 mg/L of immunoglobulin, after at least 14 days in
culture. In another embodiment, the cell can produce a virus or a
VLP (virus like particle). In yet another embodiment, the cell may
produce a desired cell product like a vitamin, a metabolite, a
glycoprotein, a carbohydrate, a lipid, or a lipoprotein. In another
embodiment, the cell itself is grown to be harvested. The media and
feed supplement compositions of the invention comprise
concentrations of cysteine and tyrosine that support maximal cell
growth for viral or VLP production, or maximal vitamin production,
or glycoprotein, or vaccine production, etc., while avoiding the
problems caused by their limited solubility.
[0026] Any cell may be cultured according to the present methods,
for instance bacterial cells, yeast cells, plant cells, insect
cells or animal cells, including mammalian cells. In one
embodiment, the animal cell for culturing according to the present
methods is an insect cell (e.g., Drosophila cells, Spodoptera cells
or Trichoplusia cells), a nematode cell (e.g., C. elegans cells) or
a mammalian cell (e.g., CHO cells, COS cells, VERO cells, BHK
cells, AE-1 cells, SP2/0 cells, L5.1 cells, PerC6, hybridoma cells,
or other human cells). In one embodiment, the cell is a CHO
cell.
[0027] Another aspect provides a method of preparing a cell culture
medium, comprising combining one or more small peptides or
dipeptides, as described herein, with a carbohydrate, such as
glucose, and at least one amino acid or salt thereof, such as
arginine, asparagine, aspartic acid, cysteine, glutamic acid,
histidine, isoleucine, lysine, methionine, phenylalanine, proline,
hydroxyproline, serine, threonine, tryptophan, tyrosine, glutamine,
and valine. In one embodiment, the one or more dipeptides are
selected from X-tyrosine, X-cysteine, tyrosine-X, or cysteine-X, or
a salt thereof, wherein X is selected from alanine, glycine,
serine, valine, proline, aspartic acid, or glutamic acid and
wherein the N-terminal amino acid of the one or dipeptides has a
free amino group. In another embodiment, the one or more dipeptides
are alanyl tyrosine and/or alanyl cysteine.
[0028] In other embodiments, the cell culture medium prepared
according to this method is concentrated as a 2.times. or greater
formulation, as discussed elsewhere in this application. In certain
embodiments, the one or more small peptides or dipeptides, such as
alanyl tyrosine and/or alanyl cysteine, are present in the cell
culture medium prepared according to this method.
[0029] For example, the concentration of small peptides or
dipeptides in some cell culture media or the concentrated media or
feeds may be about 0.5 g/L to about 30 g/L, about 0.5 g/L to about
25 g/L, about 0.5 g/L to about 20 g/L, about 0.5 g/L to about 16
g/L, about 0.5 g/L to about 10 g/L, about 0.5 g/L to about 5 g/L,
about 0.5 g/L to about 4 g/L, about 1 g/L to about 30 g/L, about 1
g/L to about 20 g/L, about 1 g/L to about 16 g/L, about 1 g/L to
about 10 g/L, about 1 g/L to about 5 g/L, about 2.5 g/L to about 30
g/L, about 2.5 g/L to about 20 g/L, about 2.5 g/L to about 16 g/L,
about 2.5 g/L to about 10 g/L, about 2.5 g/L to about 5 g/L, or
about 2.5 g/L to about 4.5 g/L, about 5 g/L to about 30 g/L, about
5 g/L to about 25 g/L, about 5 g/L to about 20 g/L, about 5 g/L to
about 16 g/L, about 5 g/L to about 10 g/L, about 5 g/L to about 5
g/L, about 5 g/L to about 4 g/L, and so on.
[0030] Another aspect provides a composition comprising the small
peptide- or dipeptide-containing cell culture medium described
herein and at least one cell, as described above. In one
embodiment, the at least one cell is a CHO cell.
[0031] Yet another aspect is directed to kits for use in the
cultivation of a cell. The kit may comprise one or more containers
containing the small peptide- or dipeptide-containing cell culture
medium described herein. The kit optionally comprises at least one
additional component selected from at least one growth factor, at
least one animal tissue extract, at least one animal organ extract,
at least one animal gland extract, at least one enzyme, at least
one protein, at least one vitamin, at least one cytokine, at least
one lipid, at least one trace element, at least one extracellular
matrix component, at least one buffer, at least one antibiotic, and
at least one viral inhibitor. The kit may also comprise one or more
cells or cell types.
[0032] Another aspect is directed to methods of producing a virus
or a VLP using the small peptide- or dipeptide-containing cell
culture media described herein. Specifically, the method comprises
(a) contacting a cell (e.g., a mammalian cell) with a virus under
conditions suitable to promote the infection of the cell by the
virus; and (b) cultivating the cell in the culture media described
herein under conditions suitable to promote the production of virus
by the cell. In one embodiment, the cell that produces the virus or
VLP is a mammalian cell, such as a CHO cell, or an insect cell, or
a plant cell, or a fungal cell. In another embodiment, a
non-mammalian virus or VLP is engineered such that it can infect a
mammalian host cell, for e.g., a human cell.
[0033] In yet another aspect, the disclosure provides methods of
producing a polypeptide, such as an immunoglobulin or a fragment
thereof, using the small peptide- or dipeptide-containing cell
culture media described herein. Specifically, the method comprises
cultivating a cell that has been genetically engineered to produce
a polypeptide in the dipeptide-containing culture media under
conditions suitable for expression of the polypeptide by the cell.
In one embodiment, the cell is a mammalian cell, such as a CHO
cell.
[0034] Therefore, one aspect of the invention is directed to a cell
culture medium, concentrated feed, or concentrated feed supplement
comprising at least one small peptide, said peptide comprising at
least two amino acids, wherein at least one of the amino acids is a
cysteine or a tyrosine. Another aspect of the invention is directed
to a cell culture medium, concentrated feed, or concentrated feed
supplement described above, wherein at least one of the remaining
amino acids of the small peptide are selected from the group
consisting of alanine, glycine, serine, valine, proline, aspartic
acid, and glutamic acid. Yet another aspect of the invention, at
least one of the remaining amino acids of the small peptide is
alanine or glycine. Another aspect of the invention is directed to
a cell culture medium, concentrated feed, or concentrated feed
supplement medium described above, wherein at least one of the
small peptides is a dipeptide selected from the group consisting of
X-tyrosine, X-cysteine, tyrosine-X, and cysteine-X, or a salt
thereof, and wherein X is selected from the group consisting of
alanine, glycine, serine, valine, proline, aspartic acid, and
glutamic acid, and/or, and wherein the N-terminal amino acid of the
one or small peptides has a free amino group. In one aspect the X
is alanine or glycine.
[0035] In some aspects of this invention, the small peptide may be
either a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a
hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a
decapeptide; or, the small peptide may be at least about two to ten
amino acids in length; or in some aspects, the small peptide is at
least about ten amino acids in length. In some aspects, the short
peptide may comprise two, three, four, five or six amino acids.
[0036] In other aspects, the cell culture medium is a liquid
solution.
[0037] In any one of the cell culture medium described above, the
cell culture medium, concentrated feed, or concentrated feed
supplement is a dry powder or a granulated dry powder.
[0038] In a further aspect, any one of the cell culture medium
described above may comprise a carbohydrate and an amino acid, or a
salt thereof. In one embodiment, the carbohydrate is a hexose. In
other embodiments, the amino acid, or salt thereof, is any one or
more of arginine, asparagine, aspartic acid, cysteine, glutamic
acid, histidine, isoleucine, lysine, methionine, phenylalanine,
proline, hydroxyproline, serine, threonine, tryptophan, tyrosine,
and valine. In a further embodiment, the media further comprises a
vitamin, a salt, a buffering agent, or an inorganic element.
[0039] In some aspects described above, the cell culture medium,
concentrated feed, or concentrated feed supplement does not contain
a lipid, a hydrolysate or a fraction thereof, or a growth factor.
In other aspects described above, the cell culture medium,
concentrated feed, or concentrated feed supplement does not contain
a protein. Thus in some preferred aspects, the cell culture medium,
concentrated feed, or reconstituted medium comprising a small
peptide may be serum-free, protein-free and/or
hydrolysate-free.
[0040] In particular aspects, the cell culture medium, concentrated
feed, or concentrated feed supplement is concentrated as a 2.times.
or greater formulation. In a preferred aspect, the cell culture
medium, concentrated feed, or concentrated feed supplement
described above comprises one or more dipeptides, which may be
either alanyl tyrosine and/or alanyl cysteine and/or alanyl cystine
dimer. In a preferred aspect, the one or more dipeptides may be
present at a concentration of about 1 g/L to about 16 g/L; or the
one or more dipeptides may be present at a concentration of about
2.5 g/L to about 8.5 g/L. In one aspect, the liquid solution stored
at 2-8.degree. C. remains free of precipitate for over 12
months.
[0041] The present invention is also directed to a method of
culturing a cell, comprising contacting the cell with a basal cell
culture medium under conditions supporting the cultivation of the
cell and supplementing the basal cell culture medium with a
concentrated feed or medium described above. The supplementation
may be done during a fed-batch culture; or performed in addition to
an existing feeding schedule; or, the supplementation may be
performed continuously as opposed to incrementally with bolus
additions. In some cases, the basal cell culture medium may be
supplemented with the concentrated feed or medium on more than one
day; or, the supplementation of the basal cell culture medium may
be done with a single concentrated feed, or with multiple
concentrated feeds; or, the supplementation may occur either from
day 0, day 1, day 2, day 3, day 4 or day 5 after starting the cell
culture, and is supplemented daily thereafter through day 13 or 14;
or, each supplementation may be about 1-10%, or about 5-20%, of the
total starting volume of the basal cell culture medium.
[0042] In all aspects of the methods used herein, the cell may be
an engineered cell; or the cell may be a recombinant cell; or the
cell may be a plant cell, or the cell may be any one of an animal,
a plant, an insect, an avian, yeast, algal or a fish cell. The
animal cell may be mammalian, insect, bovine, primate, or a
pluripotent stem cell. In one aspect, the animal cell may be a
mammalian cell; and the mammalian cell may be keratinocytes,
cervical epithelial cells, bronchial epithelial cells, tracheal
epithelial cells, kidney epithelial cells and retinal epithelial
cells) and established cell lines and their strains (e.g., 293
embryonic kidney cells, BHK cells, HeLa cervical epithelial cells
and PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK
cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562
cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS180
cells, LS174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells,
T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells,
LLC-MK.sub.2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1
cells, Y-1 cells, LLC-PK.sub.1 cells, PK(15) cells, GH.sub.1 cells,
GH.sub.3 cells, L2 cells, LLC-RC 256 cells, MH.sub.1C.sub.1 cells,
XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives
thereof), fibroblast cells from any tissue or organ (including but
not limited to heart, liver, kidney, colon, intestines, esophagus,
stomach, neural tissue (brain, spinal cord), lung, vascular tissue
(artery, vein, capillary), lymphoid tissue (lymph gland, adenoid,
tonsil, bone marrow, and blood), spleen, and fibroblast and
fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33
cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells,
Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit
529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells,
Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38
cells, WI-26 cells, MiCl.sub.1 cells, CHO cells, CV-1 cells, COS-1
cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells,
BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB
cells, BLO-11 cells, NOR-10 cells, C.sub.3H/IOTI/2 cells,
HSDM.sub.1C.sub.3 cells, KLN.sub.2O.sub.5 cells, McCoy cells, Mouse
L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells,
L-MTK.sup.- (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1
cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, C.sub.II
cells, and Jensen cells, Sp2/0, NS0, NS1 cells or engineered cells
thereof. In one aspect, the mammalian cell may be a CHO cell.
[0043] In all aspects of the methods used herein, the cells may
produce an immunoglobulin or a fragment thereof; and the cell may
produce greater than 3000 mg/L of immunoglobulin or a fragment
thereof.
[0044] Alternately, in all aspects of the methods used herein, the
cells may produce a virus or a virus-like particle (VLP); and the
virus may be a recombinant virus. The virus may be but is not
limited to an adenovirus, lentivirus, baculovirus, sendai virus,
vaccinia virus, or an engineered viral derivative thereof. In the
aspects described herein, the VLP may carry a nucleic acid, or it
may carry a RNA.
[0045] In yet another aspect, the present invention is also
directed to a method of preparing a cell culture medium,
concentrated feed or cell culture supplement, comprising combining
one or more small peptides with a carbohydrate and at least one
amino acid or salt thereof, wherein each of the one or more small
peptides comprises a cysteine or a tyrosine. In one embodiment, the
amino acids of the one or more small peptides, which are other than
cysteine or tyrosine, (or referred to as the remaining amino acids)
may be selected from the group consisting of alanine, glycine,
serine, valine, proline, aspartic acid, arginine, glutamine or
glutamic acid. In one preferred aspect, the amino acids other than
cysteine or tyrosine, are alanine or glycine. In another set, the
remaining amino acids may also be one or more of arginine,
asparagine, aspartic acid, cysteine, glutamic acid, histidine,
isoleucine, lysine, methionine, phenylalanine, proline,
hydroxyproline, serine, threonine, tryptophan, tyrosine, and
valine. In another preferred aspect of the above method, one or
more dipeptides may be selected from X-tyrosine, X-cysteine,
tyrosine-X, or cysteine-X, or a salt thereof, wherein X is selected
from alanine, glycine, serine, valine, proline, aspartic acid,
arginine or glutamic acid, and wherein the N-terminal amino acid of
the one or more dipeptide has a free amino group. In a most
preferred aspect, X is alanine or glycine. In a most preferred
aspect, the carbohydrate may be glucose. Additionally, the cell
culture medium may further comprise a vitamin, a salt, a buffering
agent, or an inorganic element; or, may not contain a lipid, a
hydrolysate or a fraction thereof, or a growth factor; or may not
contain a protein; or, the cell culture medium may be concentrated
as a 2.times. or greater formulation; or, the one or more
dipeptides may be alanyl tyrosine and/or alanyl cysteine; or, the
one or more dipeptides are present in the cell culture medium at a
concentration of about 1 g/L to about 16 g/L; or preferably, the
one or more dipeptides are present at a concentration of about 2.5
g/L to about 8.5 g/L. In most aspects, the cell culture medium
stored at 2-8.degree. C. remains free of precipitate for over 12
months.
[0046] The present invention is also directed to a method for
analyzing a cell culture medium, concentrated feed, or
reconstituted medium, the method comprising determining the
presence or absence of a short peptide comprising cysteine or
tyrosine in the medium. In one aspect, the cell culture medium,
concentrated feed, or reconstituted medium may be serum-free,
protein-free, hydrolysate-free, and the analyzing may be performed
by, but may not be limited to, mass spectrometry (LCMS); capillary
electrophoresis and HPLC.
[0047] The invention is also directed to a composition comprising
the cell culture medium, concentrated feed, or reconstituted medium
described above and a cell.
[0048] The invention is also directed to a method of making a
recombinant protein in a cell culture medium comprising: contacting
a cell with the cell culture medium, concentrated feed, or
reconstituted medium described above; and cultivating said cell
under conditions suitable for the growth of said cell and/or the
expression of said recombinant protein.
[0049] The invention is also directed to a kit for the cultivation
of a cell in vitro, said kit comprising one or more containers,
wherein a first container contains a medium comprising at least one
dipeptide described above, and wherein said medium supports the
growth of the cell in culture and/or the expression of recombinant
protein. The culturing of the cell may be in suspension or in
adherent culture. The growth may be high-density growth.
[0050] The invention is also directed to a method of making a virus
or a viral particle in a cell culture medium, concentrated feed, or
reconstituted medium described above comprising: culturing a
recombinant cell in the cell culture medium described above,
wherein the medium supports the growth of said cell under
conditions suitable for expression of said virus or viral
particle.
[0051] The invention is also directed to a recombinant protein
produced by the methods described above; or, the virus or viral
particle produced by the methods described above; or, the use of a
cell culture medium, concentrated feed, or reconstituted medium
described above, to produce a virus or a recombinant protein; or, a
culture medium, concentrated feed, or reconstituted medium produced
by the methods described above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate certain
embodiments of the invention, and together with the written
description, serve to explain certain principles of the
invention.
[0053] FIG. 1 shows viable cell density (.times.10.sup.6 cells/mL)
(depicted by curves), and IgG titer (mg/L) (depicted by bars) of
CHO cells grown in (i) a basal cell culture medium (CD
FortiCHO.TM.) supplemented with either a glucose feed, and (ii) CD
FortiCHO.TM.+aa (concentrated amino acid mixture with low levels of
cysteine and tyrosine, with glucose)+DP (dipeptide) feed schedule 2
(FP2) or (iii) CD FortiCHO.TM.+aa (concentrated amino acid mixture
with low levels of cysteine and tyrosine, with glucose)+DP
(dipeptide) feed schedule 3 (FP3). See Examples.
[0054] FIG. 2 shows an exemplary profile chromatogram of a solution
containing the Ala-Tyr dipeptide, with the Ala-Tyr dipeptide
represented by the peak at 6.172 minutes (bottom panel).
[0055] FIG. 3 shows an exemplary profile chromatogram of a solution
containing the Ala-Cys dipeptide, with the Ala-Cys dipeptide
represented by the peak at 6.631 minutes (bottom panel).
[0056] FIG. 4 shows an exemplary profile extracted ion chromatogram
of a solution containing an Ala-Tyr dipeptide standard, represented
by the peak at 8.8 minutes.
[0057] FIG. 5 shows an exemplary profile extracted ion chromatogram
of a solution containing an Ala-Cys dimer standard, represented by
the peak at 9.6 minutes.
[0058] FIG. 6 shows an exemplary profile total ion chromatogram of
an exemplary feed sample containing an Ala-Cys dimer and an Ala-Tyr
dipeptide.
[0059] FIG. 7 shows an exemplary profile extracted ion chromatogram
of an exemplary feed sample containing an Ala-Tyr dipeptide and an
Ala-Cys dimer.
DETAILED DESCRIPTION
[0060] Reference will now be made in detail to various exemplary
embodiments. It is to be understood that the following detailed
description is provided to give the reader a fuller understanding
of certain embodiments, features, and details of aspects of the
invention, and should not be interpreted as a limitation of the
scope of the invention.
[0061] 1. Definitions
[0062] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0063] As used herein, "cell culture" or "culture" refers to 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."
[0064] As used herein, "cultivation" refers to 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.
[0065] As used herein, "cell culture medium," "culture medium," or
"medium" (and in each case plural media) refer to a nutritive
composition that supports the cultivation and/or growth of cells.
The cell culture medium may be a complete formulation, i.e., a cell
culture medium that requires no supplementation to culture cells,
may be an incomplete formulation, i.e., a cell culture medium that
requires supplementation or may be a medium that may supplement an
incomplete formulation or in the case of a complete formulation,
may improve culture or culture results. The terms "cell culture
medium," "culture medium," or "medium" (and in each case plural
media) refer to unconditioned cell culture media that has not been
incubated with cells, unless indicated otherwise from the context.
As such, the terms "cell culture medium," "culture medium," or
"medium" (and in each case plural media) are distinguished from
"spent" or "conditioned" medium, which may contain many of the
original components of the medium, as well as a variety of cellular
metabolites and secreted proteins.
[0066] As used herein, "small peptide" refers to a chain of two to
six amino acids joined together by one or more peptide or
equivalent bonds, wherein at least one of the amino acids is a
tyrosine or a cysteine. Preferably the amino acids in the small
peptide, other than tyrosine or cysteine, exhibit good solubility
properties at neutral pH. Thus, in one embodiment, the amino acids
in the small peptide, other than tyrosine or cysteine, are selected
from alanine, glycine, serine, valine, proline, glutamic acid,
aspartic acid, glutamine, and arginine. In another embodiment, the
N-terminal amino acid of the small peptide has a free amino group.
In an embodiment of the invention, the small peptide may comprise a
non-amino acid with a free amine group, like ethanolamine, or a
free carboxylic group, or a similar compound.
[0067] As used herein, "extract" refers to a composition comprising
a component of a substance or 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).
[0068] As used herein, 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 can be used in cell
culture media include amino acids, salts, metals, sugars,
carbohydrates, 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.
[0069] A "1.times. formulation" refers 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 Cell Culture Technology for
Pharmaceutical and Cell-Based Therapies, 42-50 (Sadettin Ozturk and
Wei-Shou Hu eds., Taylor and Francis Group 2006), 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.
[0070] 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,
"5.times. formulation," "25.times. formulation," "50.times.
formulation," "100.times. formulation," "500.times. formulation,"
and "1000.times. formulation" designate solutions that contain
ingredients at about 5- 25-, 50-, 100-, 500-, or 1000-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. A formulation may
contain components or ingredients at 1.times. with respect to a
particular cell culture protocol, but at a concentration, for
example, 2, 2.5, 5, 6.7, 9, 12 etc. X with respect to a different
culture protocol or different base medium.
[0071] A dimer consists of two dipeptides. So, for e.g., a dimer of
Ala-Cys would comprise Ala-Cys-Cys-Ala, wherein the cys-cys are
bonded by a disulfide linkage. Ala-Cys-Cys-Ala could also be
referred to as N,N'-di-L-Alanyl-L-Cystine.
[0072] 1. Small Peptides
[0073] The present disclosure concerns the use of small peptides,
including dipeptides, in cell culture medium. Applicants have found
that preparing cell culture medium containing the desired
concentrations of certain amino acids, such as tyrosine and
cysteine, has not been possible due to the low solubility of
tyrosine and low stability of cysteine. The solubility and
stability problems of tyrosine and cysteine have been addressed by
using less than the desired concentration of tyrosine or cysteine
in the aqueous cell culture medium, thereby making them the rate
limiting amino acids for optimal cell growth and/or protein
production, as nutrients are used during cell culture. Although
using less than the desired concentration of tyrosine and cysteine
may result in an acceptable aqueous shelf life, solubility and
stability, it may also require the addition of higher volumes of
media during cell culture supplementation, for e.g., in a fed-batch
culture system to achieve desired productivity. This is
undesirable, as the stoichiometric balance, pH and/or osmolality of
the system would need to be re-adjusted as well.
[0074] The problem with large volume supplementation can be
addressed by using a concentrated feed supplement. A desirable
concentrated feed supplement is a nutritionally complex,
stoichiometrically balanced nutrient additive that supplements
nutrient-depleted culture systems, in a fed-batch culture system,
which additionally, and desirably, may have auto-pH and
auto-osmolality balancing characteristics. However, due to the low
solubility of tyrosine and low stability of cysteine, concentrated
feed compositions with high levels of cysteine and tyrosine have
been a challenge to prepare. It generally results in the cysteine
or tyrosine coming out of solution over time. Single part,
pH-neutral, chemically-defined concentrated feeds that are stable
have thus been difficult to synthesize. To circumvent the problem,
multi-part concentrated feeds that are acidic or alkaline have been
prepared.
[0075] Applicants have discovered that it is possible to substitute
the free amino acids, tyrosine and cysteine, in a cell culture
medium with small peptides like dipeptides alanyl-tyrosine,
glycyl-tyrosine, alanyl-cysteine (which forms a disulfide dimer,
[AlaCys].sub.2), or glycyl-cysteine (which forms a disulfide dimer,
[GlyCys].sub.2), or other small peptides comprising one or more
tyrosines or cysteines. Cell culture medium containing these
dipeptides, or other small peptides, provides cells with
concentrations of tyrosine and/or cysteine sufficient to support
maximal cell growth and/or protein production while avoiding the
solubility and stability problems associated with free tyrosine
and/or cysteine.
[0076] The small peptides of the invention have two to six amino
acids, wherein at least one of the amino acids is a cysteine or
tyrosine, and where the remaining amino acids can be any amino
acid. Preferably the amino acids in the small peptide, other than
tyrosine or cysteine, exhibit good solubility properties at neutral
pH. In one embodiment, the amino acids in the small peptide other
than tyrosine or cysteine are selected from alanine, glycine,
serine, valine, proline, glutamic acid, or aspartic acid. In
another embodiment, the N-terminal amino acid of the small peptide
has a free amino group. In an embodiment of the invention, the
small peptide (di, tri, four, five or six amino acids) may comprise
a non-amino acid with a free amine group, like ethanolamine, or a
free carboxylic group, or a similar compound that can contribute to
a peptide bond. In another embodiment, the small peptide may
comprise cysteine dimerized via the sulfide bond. Due to
cysteine-cysteine dimerization (cys-cys) dimerization, although the
small peptides of the invention are defined as having a length of 2
to 6 amino acids, there may be compositions that result in longer
peptides due to spontaneous cysteine-cysteine dimerization that may
occur within a media or feed of the invention. Such compounds are
also soluble, even in high concentrations, and as such, are
completely within the scope of this invention.
[0077] In other embodiments, the small peptides are dipeptides
having two amino acids and represented by the formula: X-tyrosine,
X-cysteine, tyrosine-X, or cysteine-X, or a salt thereof, wherein X
is any amino acid, and wherein the N-terminal amino acid of the
dipeptide has a free amino group. Thus, the dipeptides are distinct
from the N-acyl dipeptides disclosed in U.S. Pat. No. 5,534,538,
which require an acyl group covalently bonded to the amino group of
the N-terminal amino acid. According to U.S. Pat. No. 5,534,538, it
is the presence of the acyl group that confers the superior
stability properties to the dipeptides. Preferably, X is an amino
acid with good solubility properties. In one embodiment, X is
alanine or glycine. In another embodiment, X is serine, valine,
proline, glutamic acid, or aspartic acid.
[0078] In one embodiment, the small peptides are dipeptides having
two amino acids, and the one or more dipeptides of the invention
are selected from X-tyrosine, X-cysteine, tyrosine-X, and
cysteine-X, or a salt thereof, wherein X is any amino acid, and
wherein the N-terminal amino acid of the dipeptide has a free amino
group. In one embodiment, X is alanine or glycine. In another
embodiment, X is any amino acid, derivative of an amino acid, a
non-amino acid that has an amino-group, like ethanolamine. In an
aspect of this embodiment, the preferred amino acid is selected
from the group: serine, valine, proline, aspartic acid, arginine,
glutamine, or glutamic acid.
[0079] In another embodiment, the small peptides are tripeptides
having three amino acids, including the following tripeptides:
X-X-tyrosine, X-X-cysteine, X-tyrosine-X, X-cysteine-X,
tyrosine-X-X, cysteine-X-X, or a salt thereof, wherein X is any
amino acid. In one embodiment, the N-terminal amino acid of the
tripeptide has a free amino group. Preferably, X is an amino acid
with good solubility properties. In one embodiment, X is alanine or
glycine. In another embodiment, X is serine, valine, proline,
glutamic acid, glutamine, arginine, or aspartic acid. In a certain
aspect of the invention, the compositions described above that
containing cysteine and tyrosine in small peptides is used to
increase protein production in a cell line. In a preferred aspect
of the invention, the increased protein is an antibody that is
produced in higher titers than in media without the small peptides.
By higher antibody production is meant about 0.1 g/L to about 10
g/L, about 0.1 g/L to about 5 g/L, about 0.1 g/L to about 2.5 g/L,
about 0.1 g/L to about 1 g/L, preferably about 1 g/L to about 10
g/L, more preferably about 2 g/L to about 8 g/L, and so on.
[0080] Depending on the cell and the intended use of the cell, one
or more small peptides or dipeptides of the cell culture medium,
feed, concentrated media or feed, etc. will optimally be present at
concentrations stoichiometrically balanced to optimize cell culture
performance. For example, the concentration of one or more small
peptides or dipeptides in some cell culture media or the
concentrated feeds may be at about 0.5 g/L to about 30 g/L, about
0.5 g/L to about 25 g/L, about 0.5 g/L to about 20 g/L, about 0.5
g/L to about 16 g/L, about 0.5 g/L to about 10 g/L, about 0.5 g/L
to about 5 g/L, about 0.5 g/L to about 4 g/L, about 1 g/L to about
30 g/L, about 1 g/L to about 20 g/L, about 1 g/L to about 16 g/L,
about 1 g/L to about 10 g/L, about 1 g/L to about 5 g/L, about 2.5
g/L to about 30 g/L, about 2.5 g/L to about 20 g/L, about 2.5 g/L
to about 16 g/L, about 2.5 g/L to about 10 g/L, about 2.5 g/L to
about 5 g/L, or about 2.5 g/L to about 4.5 g/L, about 5 g/L to
about 30 g/L, about 5 g/L to about 25 g/L, about 5 g/L to about 20
g/L, about 5 g/L to about 16 g/L, about 5 g/L to about 10 g/L,
about 5 g/L to about 5 g/L, about 5 g/L to about 4 g/L, and so on.
In some media formulations or prototype media formulations, one or
more small peptides, and/or one or more dipeptides may be present
at a concentration of about 2.5 g/L to about 8.5 g/L in the media
or prototype media. Additionally, some embodiments of the invention
provide methods of preparing "auto-pH media" or feed powders which
automatically are at a desired pH upon rehydration/reconstitution
with a solvent. In accordance with the invention, such media or
feeds may be in powdered (dry powder (DPM), advanced powder (APM)
or advanced granulating technology (AGT) or in liquid form.
[0081] Animal cell culture media or feeds prepared by the present
methods will, upon reconstitution, preferably have a pH of about
6-8, or about 7-8, or about 6.0-6.3 (for insect cells), more
preferably about 7-7.5 or about 7.2-7.4, most preferably around
7.0; and plant cell culture media or feeds 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, or preferably about 6.0
to 6.3. 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.
[0082] 2. Cell Culture Medium
[0083] A cell culture medium is composed of a number of ingredients
and these ingredients vary from one culture medium to another. As
noted above, a cell culture medium may be a complete formulation,
i.e., a cell culture medium that requires no supplementation to
culture cells, may be an incomplete formulation, i.e., a cell
culture medium that requires supplementation or may be a supplement
that may supplement an incomplete formulation or in the case of a
complete formulation, may improve culture or culture results.
[0084] Generally a cell culture medium will have solutes dissolved
in solvent. The solutes provide an osmotic force to balance the
osmotic pressure across the cell membrane (or wall). Additionally
the solutes will provide nutrients for the cell. Some nutrients
will be chemical fuel for cellular operations; some nutrients may
be raw materials for the cell to use in anabolism; some nutrients
may be machinery, such as enzymes or carriers that facilitate
cellular metabolism; some nutrients may be binding agents that bind
and buffer ingredients for cell use or that bind or sequester
deleterious cell products.
[0085] Depending on the cell and the intended use of the cell, the
ingredients of the cell culture medium will optimally be present at
concentrations balanced to optimize cell culture performance.
Performance will be measured in accordance with a one or more
desired characteristics, for example, cell number, cell mass, cell
density, O.sub.2 consumption, consumption of a culture ingredient,
such as glucose or a nucleotide, production of a biomolecule,
secretion of a biomolecule, formation of a waste product or by
product, e.g., a metabolite, activity on an indicator or signal
molecule, etc. Each or a selection of the ingredients will thus
preferably optimized to a working concentration for the intended
purpose.
[0086] Culture media or feed supplements of the invention may be
available in a dry format that requires only addition of a solvent
such as water. Preferably the dry format powder is prepared by at
least one method selected from the group consisting of milling,
impacting, extruding and cutting or breaking, wet granulation, high
shear granulation, pan granulation and fluidized bed agglomeration.
Dry formats include, but are not limited to, dry powder format
(DPM), agglomerated (AGT.TM.) format, advanced powder media (APM),
or other suitable dry formats. Preferably, once water is added,
dissolution should occur quickly and the resultant liquid can be
filtered and added directly to the cells without any pH adjustment.
The reconstituted medium or concentrated supplement may be prepared
in variable bulk quantities and is amenable to sterilization,
particularly by ionizing or ultraviolet irradiation.
[0087] a. Carbohydrates
[0088] Cell culture medium ingredients typically include a
carbohydrate, amino acids, salts, trace elements, and vitamins. For
mammalian cells, the main carbohydrate used in cell culture media
is glucose, routinely supplemented at 5 to 25 nM. See Cell Culture
Technology for Pharmaceutical and Cell-Based Therapies, 51
(Sadettin Ozturk and Wei-Shou Hu eds., Taylor and Francis Group
2006). In addition to glucose, any hexose like galactose, fructose,
or mannose or a combination of these may be used. In addition,
mammalian cells can also use glutamine as a major energy source.
Glutamine is often included at higher concentrations than other
amino acids (2-8 mM). However, as noted above, glutamine can
spontaneously break down to form ammonia and certain cell lines
produce ammonia faster, which is toxic. Therefore, glutamate and
glutamine dipeptides have been used as substitutes for glutamine to
reduce the build up of toxic ammonia in the cell culture
medium.
[0089] b. Amino Acids
[0090] Amino acids are important in cell culture medium for
maintaining the metabolic function of the cultured cells. Cell
culture medium typically includes the essential amino acids (i.e.,
those amino acids that are normally not synthesized in vivo by
mammals) as well as certain non-essential amino acids. A non
essential amino acid is typically included in the cell culture
medium if the cell line is not capable of synthesizing the amino
acid or if the cell line cannot produce sufficient quantities of
the amino acid to support maximal growth. Exemplary amino acids
include L-alanine, L-arginine, L-asparagine, L-aspartic acid,
L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine,
L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine,
L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan,
L-tyrosine, and L-valine.
[0091] c. Salts
[0092] Salts are added to cell culture medium to maintain isotonic
conditions and prevent osmotic imbalances. The osmolality of
standard mammalian cell culture medium is about 300 mOsm/kg,
although many cell lines can tolerate an approximately 10%
variation of this value. The osmolality of some insect cell
cultures tend to be higher than 300 mOsm/kg, and this may be 0.5%,
1%, 2 to 5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30% higher than 300
mOsm/kg. The most commonly used salts in cell culture medium
include Na.sup.+, K.sup.+, Mg.sup.2+, Ca.sup.2+, Cl.sup.-,
SO.sub.4.sup.2-, PO.sub.4.sup.3-, and HCO.sub.3.sup.- (e.g.,
CaCl.sub.2, KCl, NaCl, NaHCO.sub.3, Na.sub.2HPO.sub.4). Therefore,
the desired osmolality for a cell culture medium for cultivation of
a particular cell type may also be determined empirically by one of
ordinary skill in the art, using art-known methods.
[0093] d. Inorganic Elements
[0094] Other inorganic elements that are present in serum in trace
amounts can be included in cell culture medium, as described in US
2005/0287666, which is hereby incorporated by reference in its
entirety. They include Mn, Cu, Zn, Mo, Va, Se, Fe, Ca, Mg, Si, and
Ni. Other inorganic elements that have been added to cell culture
medium, although not as frequently, include Al, Ag, Ba, Br, Cd, Co,
Cr, F, Ge, J, Rb, and Zr. Many of these elements are involved in
enzymatic activity. They may be provided in the form of salts such
as CaCl.sub.2, Fe(N03).sub.3, MgCl.sub.2, MgSO.sub.4, MnCl.sub.2,
NaCl, NaHCO3, Na2HPO4, and ions of the trace elements, such as,
selenium, vanadium and zinc. These trace elements may be provided
in a variety of forms, preferably in the form of salts such as
Na.sub.2SeO.sub.3, NH.sub.4VO.sub.3, etc. These inorganic salts and
trace elements may be obtained commercially, for example from Sigma
(Saint Louis, Mo.).
[0095] e. Vitamins
[0096] Vitamins are typically used by cells as cofactors. The
vitamin requirements of each cell line vary greatly, although
generally extra vitamins are needed if the cell culture medium
contains little or no serum or if the cells are grown at high
density. Exemplary vitamins include biotin, choline chloride, folic
acid, i-inositol, nicotinamide, D-Ca.sup.++-pantothenate,
pyridoxal, riboflavin, thiamine, pyridoxine, niacinamide, A,
B.sub.6, B.sub.12, C, D.sub.3, E, K, and p-aminobenzoic acid
(PABA).
[0097] f. Serum
[0098] Serum, the supernatant of clotted blood, can be used in cell
culture medium to provide components that promote cell growth
and/or productivity. These serum components include attachment
factors, micronutrients (e.g., trace elements), growth factors
(e.g., hormones, proteases), and protective elements (e.g.,
antitoxins, antioxidants, antiproteases). Serum is available from a
variety of animal sources including bovine or equine. When included
in cell culture medium, serum is typically added at a concentration
of 5-10%. Certain cell culture media are serum free.
[0099] g. Growth Factors
[0100] To promote cell growth in the absence or serum or in serum
reduced media, one or more of the following polypeptides can be
added to a cell culture medium: for example, fibroblast growth
factor (FGF), including acidic FGF and basic FGF, insulin,
insulin-like growth factor (IGF), epithelial growth factor (EGF),
nerve growth factor (NGF), platelet-derived growth factor (PDGF),
and transforming growth factor (TGF), including TGF.alpha. and
TGF.beta., any cytokine, such as interleukins 1, 2, 6, granulocyte
stimulating factor, Leukocyte inhibitory factor (LIF), etc.
[0101] In certain embodiments, the cell culture medium does not
contain a growth factor. In protein-free media, insulin may be
replaced with zinc or a zinc containing compound, as described in
WO 98/08934, which is hereby incorporated by reference in its
entirety.
[0102] h. Lipids
[0103] One or more lipids can also be added to a cell culture
medium. Serum typically contains lipids, such as fatty acids (e.g.,
linoleic acid, linolenic acid, arachidonic acid, palmitoleic acid,
oleic acid, polyenoic acid, and/or fatty acids of 12, 14, 16, 18,
20, or 24 carbon atoms, each carbon atom branched or unbranched),
phospholipids, lecithin (phophatidylcholine), and cholesterol.
Alternatively, one or more of these lipids can be included as
supplements in serum-free media. Phosphatidic acid and
lysophosphatidic acid stimulate the growth of certain
anchorage-dependent cells, such as MDCK, mouse epithelial, and
other kidney cell lines, while phosphatidylcholine,
phosphatidylethanolamine, and phosphatidylinositol stimulate the
growth of human fibroblasts in serum-free media. Ethanolamine and
cholesterol have also been shown to promote the growth of certain
cell lines. In certain embodiment, the cell culture medium does not
contain a lipid.
[0104] i. Carrier Proteins
[0105] One or more carrier proteins, such as bovine serum albumin
(BSA) or transferrin, can also be added to the cell culture medium.
Carrier proteins can help in the transport of certain nutrients or
trace elements. BSA is typically used as a carrier of lipids, such
as linoleic and oleic acids, which are insoluble in aqueous
solution. In addition, BSA can also serve as a carrier for certain
metals, such as Fe, Cu, and Ni. In protein-free formulations,
non-animal derived substitutes for BSA, such as cyclodextrin, can
be used as lipid carriers. Transferrin is involved in transporting
iron across cell membranes. In certain cases, human serum albumin
may be necessary for the cultivation of cells (for. e.g., such as
in xeno-free (XF) culture) desirable for products generated for
downstream therapeutic use. In other instances, recombinant human
serum albumin may be used in the cell culture medium for the
cultivation of cells. In particular cases, the recombinant human
serum albumin may be derived from plant, algal or fungal sources
such as rice, corn, potato, wheat, even yeast, etc. to provide for
animal-origin free (AOF) culturing of cells. In protein-free
formulations, transferrin can be replaced by ferric and/or ferrous
salts, as described in WO 98/08934, which is hereby incorporated by
reference in its entirety, or a hydroxypyridine derivative, as
described in US 2007/0254358, which is hereby incorporated by
reference in its entirety. Additionally, in protein-free
formulations, insulin can be replaced by zinc, vanadium or other
suitable divalent salts.
[0106] j. Attachments Proteins
[0107] One or more attachment proteins, such as fibronectin,
laminin, and pronectin, can also be added to a cell culture medium
to help promote the attachment of anchorage-dependent cells to a
substrate.
[0108] k. Buffering Agent
[0109] The cell culture medium can optionally include one or more
buffering agents. Suitable buffering agents include, but are not
limited to, N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic
acid] (HEPES), MOPS, MES, phosphate, bicarbonate and other
buffering agents suitable for use in cell culture applications. A
suitable buffering agent is one that provides buffering capacity
without substantial cytotoxicity to the cells cultured. The
selection of suitable buffering agents is within the ambit of
ordinary skill in the art of cell culture.
[0110] 1. Polyanionic or Polycationic Compounds
[0111] Polyanionic or polycationic compounds can prevent the cells
from clumping and promote growth of the cells in suspension. See WO
98/08934, which is hereby incorporated by reference in its
entirety. Exemplary polyanionic compounds include polysulfonated or
polysulfated compound, such as, heparin, dextran sulfate, heparan
sulfate, dermatan sulfate, chondroitin sulfate, pentosan
polysulfate, a proteoglycan or the like.
[0112] In addition to the small peptides or dipeptides described
herein, the cell culture medium comprises one or more ingredients,
such as those listed above. In one embodiment, the cell culture
medium comprises one or more small peptides or one or more
dipeptides, as described herein, and optionally comprises one or
more of the following ingredients: ethanolamine, D-glucose, HEPES,
insulin, a cytokine (e.g., IL-6), heparin, dextran sulfate,
linoleic acid, lipoic acid, phenol red, PLURONIC.RTM. F68,
putrescine, sodium pyruvate, transferrin, L-alanine, L-arginine,
L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid,
L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine,
L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,
L-threonine, L-tryptophan, L-tyrosine, L-valine, biotin, choline
chloride, D-Ca.sup.++-pantothenate, folic acid, i-inositol,
niacinamide, pyridoxine, riboflavin, thiamine, vitamin B.sub.12,
one or more calcium salts, Fe(NO.sub.3).sub.3, KCl, one or more
magnesium salts, one or more manganese salts, NaCl, NaHCO.sub.3,
Na.sub.2HPO.sub.4, one or more selenium salts, one or more vanadium
salts and one or more zinc salts. In one embodiment, the one or
more dipeptides are selected from X-tyrosine, X-cysteine,
tyrosine-X, and cysteine-X, or a salt thereof, wherein X is any
amino acid, and wherein the N-terminal amino acid of the dipeptide
has a free amino group. In one embodiment, X is alanine or glycine.
In another embodiment X is serine, valine, proline, aspartic acid,
or glutamic acid.
[0113] The media described herein can be a 1.times. formulation or
can be concentrated as anything greater than a 1.times.
formulation, for example, as a 2.times., 5.times., 10.times.,
20.times., 50.times., 500.times., or 1000.times. medium formulation
as the solubilities of the individual components allow. If the
individual medium ingredients are prepared as separate concentrated
solutions, an appropriate (sufficient) amount of each concentrate
is combined with a diluent to produce a 1.times. medium
formulation. Typically, the diluent used is water but other
solutions including aqueous buffers, aqueous saline solution, or
other aqueous solutions may be used.
[0114] The media described herein can also be prepared in different
forms, such as dry powder media, a granulated preparation (which
requires addition of water, but not other processing, such as
pHing), liquid media or as media concentrates.
[0115] 3. Serum-Free Medium
[0116] Potential problems associated with serum, including batch to
batch variation, high protein content, risk of contaminants (e.g.,
viruses, mycoplasma, prions), limited availability, and high cost,
have driven the production of serum-free media. Furthermore,
improved levels of recombinant protein expression can be obtained
from cells grown in serum-free medium, relative to the level of
expression seen in cells grown in medium supplemented with serum
(Battista, P. J. et al., Am. Biotech Lab. 12: 64-68 (1994)).
[0117] In these serum-free media, serum can be replaced with a
defined hormone, or hormone cocktails, such as HITES or ITES, which
contain hydrocortisone, insulin, transferrin, ethanolamine, and
selenite. Alternatively, the serum-free media can contain growth
factor extracts from endocrine glands, such as epidermal or
fibroblast growth factors. Serum-free media can also contain other
components as a substitute for serum, including purified proteins
(animal or recombinant), peptones, amino acids, inorganic salts,
and animal or plant hydrolysates (or fractions thereof).
[0118] Serum-free media may be chemically defined or undefined. In
chemically defined media, the identity of the components and their
amounts are known, whereas the opposite is true for chemically
undefined media. Chemically defined media, therefore, are designed,
in part, to reduce the risk of contaminants and to reduce batch to
batch variation. Chemically defined supplements that can be added
to cell culture media include growth factors, hormones, carrier
proteins, and/or attachment factors. In a preferred embodiment, the
basal medium used with the media or feeds comprising small peptides
is a chemically-defined medium. In another preferred embodiment,
the concentrated cell culture media or concentrated feed of the
invention comprising small peptides is also a chemically-defined
composition. In yet another preferred embodiment, the concentrated
feed or medium of the invention comprising small peptides including
cysteine and tyrosine is a single part feed and is
chemically-defined. In another aspect, all the above compositions
comprising small peptides including cysteine and tyrosine are
auto-pH and auto-osmolality balanced. In yet another aspect, all
the above compositions comprising small peptides including cysteine
and tyrosine are stoichiometrically balanced.
[0119] 4. Protein-Free Media
[0120] Serum-free media contains reduced amounts of protein as
compared to cell culture media containing serum. However,
serum-free media may still contain one or more of a variety of
animal-derived components, including albumin, fetuin, various
hormones and other proteins. The presence of proteins makes
purification of recombinant protein difficult, time-consuming, and
expensive and can also lead to reduced product yields and/or
purity. Thus, in one embodiment, the cell culture medium is protein
free.
[0121] Protein-free media can be obtained by methods known in the
art, such as by removing any remaining proteins from serum-free
media. While the removal of such proteins from the cell culture
media can impair the media's ability to support cell growth, other
components can be added to the media to mitigate the effect of
removing the proteins from the media. For example, as discussed
above, cyclodextrin can replace BSA and iron salts or a
hydroxypyridine derivative can replace transferrin. In other cases,
animal tissue or plant hydrolysates (or fractions thereof) have
been used to supplement protein-free media.
[0122] 5. Fed-Batch Cultivation
[0123] Fed-batch cultivation of cells is typically used for
industrial production of biomolecules, such as proteins, to
increase cell concentration and to extend culture lifetime for a
high product concentration and volumetric productivity. Fed-batch
cultures involve the controlled addition of one or more nutrients,
in the form of feeds which may contain nutrients that are quickly
utilized by cells such as glucose, amino acids to a basal medium.
The nutrient(s) help to control the growth of the cell culture by
attempting to prevent nutrient depletion and byproduct
accumulation, important parameters, such as pH, osmolality and
CO.sub.2 concentration, within levels that promote cell growth or
minimize cell death for optimal product expression. See Cell
Culture Technology for Pharmaceutical and Cell-Based Therapies,
349-386 (Sadettin Ozturk and Wei-Shou Hu eds., Taylor and Francis
Group 2006). Even then, fed batch cultures often result in high
concentrations of inhibitory metabolites and high osmolalities that
eventually are incompatible with cell viability.
[0124] A basal medium is typically used for maintenance of a cell
culture, and can comprise a number of ingredients, including amino
acids, vitamins, organic and inorganic salts, sugars and other
components, each ingredient being present in an amount which
supports the cultivation of the cell in vitro. Basal media useful
for prokaryotic cell culture including bacterial and archebacterial
cultures, viral cultures, plant cell culture, insect cell culture,
mammalian cell culture can be used with the small peptide Examples
of basal media include Eagle's basal medium (BME), Eagle's minimal
essential medium (EMEM), Dulbecco's modification of Eagle's medium
(DMEM), Glasgow's modification of Eagle's medium (GMEM), Joklik's
modified Eagle's medium, Alpha modified Eagle's medium, Roswell
Park Memorial Institute (RPMI) medium, Fischer's medium, Leibovitz
L-15 medium, Trowell's T-8 medium, Williams' medium E, Biggers'
medium, Connaught Medical Research Laboratories (CMRL) 1066 medium,
Ham's F10 medium, Ham's F12 medium, Iscove's modified Dulbecco's
medium (IMDM), MCDB 104, MCDB 110, MCDB 153, Medium 199, NCTC 135
medium, and Waymouth's medium MB 752/1. For CHO cells, preferred
basal media include CD CHO, CD OptiCHO.TM., and CD FortiCHO.TM.
(all from Life Technologies, Corp., Carlsbad, Calif.). Preferred
concentrated feed supplements for CHO cells include, but are not
limited to, CHO CD EfficientFeed.TM. A (Invitrogen Cat. No.
A1023401), CHO CD EfficientFeed.TM. B (Invitrogen Cat. No.
A1024001), CHO CD EfficientFeed.TM. kit (Invitrogen Cat. No.
A1024101), CD EfficientFeed.TM. C AGT.TM. (Invitrogen Cat. No.
A1327501, Life Technologies Corp., Carlsbad, Calif.).
[0125] In fed-batch cultivation, cells are typically grown up to a
certain time point in batch mode using a basal medium.
Subsequently, a medium supplement (concentrated feed) comprising
concentrated solutions of a single or multiple nutrients is added
to provide nutrients, while minimizing volume increase or culture
dilution. When the medium supplement is added to basal medium, it
improves cell culture, as exhibited, for example, by more rapid
cell growth, decreased doubling time, higher achievable density of
cells, or higher production or yield of biomolecule, such as
protein, e.g., antibody or other proteins of therapeutic
interest.
[0126] A cell culture medium or concentrated feed of the invention,
suitable for use in supplementing a basal medium, comprises one or
more small peptides, including one or more dipeptides, as described
herein. The cell culture medium or concentrated feed comprising
cysteine and tyrosine may be optionally used in conjunction with
another feed, say, a concentrated mixture of amino acids that may
comprise one or more of the following ingredients: adenine,
ethanolamine, a carbohydrate source (such as a hexose like either
glucose, mannose, galactose, fructose, or even a combination
thereof), heparin, a buffering agent, hydrocortisone, lipoic acid,
phenol red, phosphoethanolamine, putrescine, sodium pyruvate,
tri-iodothyronine, thymidine, L-alanine, L-arginine, L-asparagine,
L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine,
L-histidine, L-hydroxyproline, L-isoleucine, L-leucine, L-lysine,
L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,
L-tryptophan, L-tyrosine, L-valine, N-acetyl-cysteine, biotin,
choline chloride, D-Ca.sup.++-pantothenate, folic acid, i-inositol,
niacinamide, pyridoxine, riboflavin, thiamine, vitamin B.sub.12,
Pluronic F68, recombinant insulin, a calcium salt, CuSO.sub.4,
FeSO.sub.4, FeCl.sub.3, Fe(NO.sub.3).sub.3, KCl, a magnesium salt,
a manganese salt, sodium acetate, NaCl, NaHCO.sub.3,
Na.sub.2HPO.sub.4, Na.sub.2SO.sub.4, a selenium salt, a silicon
salt, a molybdenum salt, a vanadium salt, a nickel salt, a tin
salt, ZnCl.sub.2, ZnSO.sub.4 or other zinc salts.
[0127] Thus, the concentrated mixture of amino acids used to
supplement the basal medium may be made up of a stoichiometrically
balanced mixture of amino acids, may or may not contain cysteine
and tyrosine, and may optionally contain one or more of the
following: a carbon source, vitamins, trace elements, and further,
may be chemically-defined in that it does not contain any lipids,
hydrolysates, or growth factors. In some instances, the
concentrated mixture of amino acids may contain animal origin free
hydrolysates or a fraction of a hydrolysate, for e.g., a plant
hydrolysate. Any commercially available concentrated mixture of
amino acids can be used, for instance, CHO CD EfficientFeed.TM. A
(Invitrogen Cat. No. A1023401), CHO CD EfficientFeed.TM. B
(Invitrogen Cat. No. A1024001), CHO CD EfficientFeed.TM. kit
(Invitrogen Cat. No. A1024101), CD EfficientFeed.TM. C AGT.TM.
(Invitrogen Cat. No. A1327501, Life Technologies Corp., Carlsbad,
Calif.).
[0128] In one embodiment, the cysteine and tyrosine containing cell
culture medium, or concentrated feed of the invention, suitable for
use in supplementing a basal medium, is protein free. In another
embodiment the cell culture medium, or concentrated feed of the
invention suitable for use in supplementing a basal medium, is
protein free and additionally does not contain lipids, hydrolysates
or a fraction thereof, or growth factors, and therefore would be
considered chemically-defined (CD). In one aspect of this
embodiment, the cysteine and tyrosine containing cell culture
medium, or concentrated feed of the invention may optionally
contain one or more of the following: a carbon source, vitamins,
trace elements, and further, may be chemically-defined in that it
does not contain any lipids, hydrolysates, or growth factors.
[0129] 6. Cells
[0130] Media containing the cysteine and tyrosine containing small
peptides or dipeptides described herein can also be used to culture
a variety of cells. Cells grown using the culture medium and feeds
described herein can be derived from any prokaryote including
archebacteria, algae, yeast, fungus, plant, insect, animal,
preferably a mammal, and most preferably a mouse or a human. In one
embodiment, the media is used to culture eukaryotic cells,
including plant or animal cells, such as mammalian cells, fish
cells, insect cells, amphibian cells or avian cells.
[0131] Mammalian cells that can be cultured with the media
described herein include primary epithelial cells (e.g.,
keratinocytes, cervical epithelial cells, bronchial epithelial
cells, tracheal epithelial cells, kidney epithelial cells and
retinal epithelial cells) and established cell lines and their
strains (e.g., 293 embryonic kidney cells, BHK cells, HeLa cervical
epithelial cells and PER-C6 retinal cells, MDBK (NBL-1) cells, 911
cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells,
Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB
cells, LS180 cells, LS174T cells, NCI-H-548 cells, RPMI 2650 cells,
SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I
cells, LLC-MK.sub.2 cells, Clone M-3 cells, 1-10 cells, RAG cells,
TCMK-1 cells, Y-1 cells, LLC-PK.sub.1 cells, PK(15) cells, GH.sub.1
cells, GH.sub.3 cells, L2 cells, LLC-RC 256 cells, MH.sub.1C.sub.1
cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or
derivatives thereof), fibroblast cells from any tissue or organ
(including but not limited to heart, liver, kidney, colon,
intestines, esophagus, stomach, neural tissue (brain, spinal cord),
lung, vascular tissue (artery, vein, capillary), lymphoid tissue
(lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, and
fibroblast and fibroblast-like cell lines (e.g., CHO cells, TRG-2
cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells,
Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525
cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells,
Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells,
MRC-5 cells, WI-38 cells, WI-26 cells, MiCl.sub.1 cells, CHO cells,
CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells,
DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells,
M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells,
C.sub.3H/IOTI/2 cells, HSDM.sub.1C.sub.3 cells, KLN.sub.2O.sub.5
cells, McCoy cells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M
strain (Mouse L) cells, L-MTK.sup.- (Mouse L) cells, NCTC clones
2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian muntjac
cells, SIRC cells, C.sub.II cells, and Jensen cells, Sp2/0, NSO, NS
1 cells or derivatives thereof).
[0132] Cells cultured according to the methods disclosed herein may
be normal cells, diseased cells, transformed cells, mutant cells,
somatic cells, genetically engineered cells, germ cells, stem
cells, precursor cells or embryonic cells, any of which may be
established or transformed cell lines or obtained from natural
sources. Cells may be used for experimental purposes or for
production of useful components. In certain cases, the cultivated
cells themselves are the products and use of cells in cellular
therapies. The cells may also be cultivated for protein production
including antibody production, for small RNA production (like miRNA
or siRNA), for virus or VLP (virus-like particle) production,
generation and isolation of DNA or viral vectors, for nucleic acid
production, for vitamin production, for desirable metabolites,
biofuel synthesis, etc. In one embodiment, the media described
herein is used to culture Chinese Hamster Ovary (CHO) cells. CHO
cells have been classified as both epithelial and fibroblast cells
derived from the Chinese hamster ovary. A cell line started from
Chinese hamster ovary (CHO-K1) (Kao, F.- T. And Puck, T. T., Proc.
Natl. Acad. Sci. USA 60: 1275-1281 (1968) has been in culture for
many years. Most biopharmaceuticals currently produce proteins in
CHO cells for many advantages that the cell line has precise
post-translation modification such as human-like glycosylation
patterns, and low risk for transmission of human viruses.
[0133] 7. Cultivation of Cells
[0134] Cells supported by the culture medium described herein can
be cultivated according to the experimental conditions determined
by the investigator. The examples below demonstrate at least one
functional set of culture conditions useful for cultivation of
certain mammalian cells. It is to be understood, however, that the
optimal plating and culture conditions for a given animal cell type
can be determined by one of ordinary skill in the art using only
routine experimentation. For routine monolayer culture conditions,
using the cell culture media described herein, cells can be plated
onto the surface of culture vessels without attachment factors.
Alternatively, the vessels can be precoated with natural,
recombinant or synthetic attachment factors or peptide fragments
(e. g., collagen, fibronectin, vitronectin, laminin and the like,
or natural or synthetic fragments thereof), which are available
commercially for example from Life Technologies, Corp. (Carlsbad,
Calif.), R&D Systems, Inc. (Rochester, Minn.), Genzyme
(Cambridge, Mass.) and Sigma (St. Louis, Mo.). Cells can also be
seeded into or onto a natural or synthetic three-dimensional
support matrix such as a preformed collagen gel or a synthetic
biopolymeric material. For suspension cultivation, cells are
typically suspended in the culture media described herein and
introduced into a culture vessel that facilitates cultivation of
the cells in suspension, such as a spinner flask, perfusion
apparatus, or bioreactor. See Cell Culture Technology for
Pharmaceutical and Cell-Based Therapies, 156-174 (Sadettin Ozturk
and Wei-Shou Hu eds., Taylor and Francis Group 2006). In some
cases, some level of agitation of the media and the suspended cells
is necessary. Agitation may be minimized to avoid denaturation of
media components and shearing of the cells during cultivation.
[0135] The cell seeding densities for each experimental condition
can be optimized for the specific culture conditions being used.
For routine monolayer culture in plastic culture vessels, an
initial seeding density of 1-5.times.10.sup.5 cells/cm.sup.2 may be
preferable, while for suspension cultivation, a higher seeding
density (e. g., 5-20.times.10.sup.5 cells/ml) may be used.
[0136] Mammalian cells are typically cultivated in a cell
incubator, preferably at about 37.degree. C., but it could be
anywhere between 30.degree. C. to 39.degree. C. Non-mammalian cells
may have other preferred temperatures for cultivation. Cultivation
of mammalian and non-mammalian cells can be done in steps, for
instance, at one temperature for optimal cell growth, and at
another temperature for optimal protein/peptide/fragment or virus
production. The incubator atmosphere may be humidified and may
contain about 3-10% carbon dioxide in air, more preferably about
5-10% carbon dioxide in air, and most preferably about 3-8% carbon
dioxide in air, although cultivation of certain cell lines may
require as much as 20% carbon dioxide in air for optimal results.
Culture medium pH may be in a preferred range depending on the cell
type, for example of about 6-8.5, preferably about 7.1 to 7.6, or
preferably about 7.1 to 7.4, or more preferably about 7.1 to 7.3 or
preferably about 6-6.3 for insect cells.
[0137] Cells in closed or batch culture should undergo complete
medium exchange (i. e., replacing spent media with fresh media)
when the cells reach a density of about 1.5-2.0.times.10.sup.6
cells/ml. Cells in perfusion culture (e. g., in bioreactors or
fermenters) will receive fresh media on a continuously
recirculating basis.
[0138] 8. Virus Production
[0139] In addition to cultivation of cells in suspension or in
monolayer cultures, the present media may be used in methods for
producing viruses from mammalian cells. Such methods comprise (a)
contacting a cell (e.g., a mammalian cell) with a virus under
conditions suitable to promote the infection of the cell by the
virus; and (b) cultivating the cell in the small peptide- or
dipeptide-containing cell culture media described herein under
conditions suitable to promote the production of virus by the cell.
The cell may be contacted with the virus either prior to, during or
following cultivation of the cell in the culture media. Optimal
methods for infecting a mammalian cell with a virus are well-known
in the art and will be familiar to one of ordinary skill
Virus-infected mammalian cells cultivated in the culture media
described herein may be expected to produce higher virus titers (e.
g., 2-, 3-, 5-, 10-, 20-, 25-, 50-, 100-, 250-, 500-, or 1000-fold
higher titers) than cells cultivated in a cell culture media other
than the cell culture media described herein.
[0140] These methods may be used to produce a variety of mammalian
viruses or viruses adapted to infect mammalian cells, viral like
particles and viral vectors, including but not limited to
adenoviruses and derivatives thereof, adeno-associated viruses and
derivatives thereof, retroviruses and derivatives thereof,
lentiviruses and derivatives thereof, insect viruses like
baculoviruses and derivatives thereof, sendai virus and derivatives
thereof, and so on. Following cultivation of the infected cells in
the culture media described herein, the used culture media
comprising viruses, which may be a recombinant virus, viral
vectors, viral particles or components thereof (proteins and/or
nucleic acids (DNA and/or RNA)) may be used for a variety of
purposes, including vaccine production, production of inhibitory
RNA molecules like miRNA, siRNA, etc., production of viral vectors
for use in cell transfection or gene therapy, infection of animals
or cell cultures, study of viral proteins and/or nucleic acids and
the like. Alternatively, viruses, viral vectors, viral particles or
components thereof may optionally be isolated from the used culture
medium according to techniques for protein and/or nucleic acid
isolation that will be familiar to one of ordinary skill in the
art.
[0141] In one embodiment, the cells produce VLPs. "VLPs" or
"virus-like particles" are vehicles for delivering one or more
compounds, including biological materials such as lipids,
carbohydrates, proteins and nucleic acids into cells. Other
compounds which may be delivered with VLPs include dyes (e.g.,
fluorescent dyes), labels (e.g., fluorescent or radioactive
labels), and drugs (e.g., antibiotics or anti-viral agents). VLPs
generally contain at least one viral protein. Typically, a viral
protein surrounds the compounds. However, in particular instances,
compounds to be delivered can be associated with a VLP by means
other than by inclusion in the VLP. For example, compounds may be
attached (e.g., covalently or non-covalently attached) to a viral
protein, or can be integrated into the envelope, when present. In
one aspect, VLPs may be associated with various types of nucleic
acids (e.g., heterologous nucleic acids) such as DNA, RNA, both RNA
and DNA, or RNA/DNA hybrids, or derivatives known in the art.
Examples of VLPs include viral particle products produced by using
VIRAPOWER.TM. adenoviral and lentiviral vector kits (see, e.g.,
Invitrogen Corporation, cat. nos. K4930-00, K4940-00, K4950-00,
K4955-00, K4960-00, K4965-00, K4967-00, and K4985-00).
[0142] Viruses which may be used to prepare VLPs include, for
examples, phage, (e.g., T even phages (e.g., T4 phage, etc.), T odd
phage (e.g., T7 phage, etc.), bacteriophage phi29, lambda phage,
etc.), baculoviruses, adenoviruses, adeno-associated viruses,
lentiviruses (e.g., Moloney Murine leukemia virus, HIV1, HTLV-III,
etc.), sendai viruses, pox viruses, and alphaviruses (e.g., Semliki
Forest Virus, SindBis Virus, etc.). Additional examples of viruses
which may be used to prepare VLPs, as well as methods for preparing
VLPs are described elsewhere herein.
[0143] 9. Recombinant Protein Production
[0144] The present culture media may also be used in methods for
the production of recombinant proteins from cells described above,
preferably mammalian cells, and particularly from mammalian cells
grown in suspension. Because the present culture media provide for
rapid, high-density suspension cultivation of mammalian cells, the
present methods facilitate enhanced production of recombinant
proteins. By protein is meant, either, a full-length protein, a
protein fragment, a peptide, a cleaved protein product, a
cross-linked protein product, tagged-peptide or proteins, etc. By
protein is also meant all types of natural and altered proteins,
including recombinant, mutant, engineered, chimeric, glycoproteins,
lipoproteins, active, processed proteins, etc. The protein may be
naturally expressed by the cell or cell line, or the cell line may
be engineered to express it using standard genetic engineering
methods known in the art including, but not limited to,
transfection, transduction, electroporation, etc. The resulting
protein or peptide may be purified or isolated to a desired level
of purity. The proteins or peptides or their fragments, that can be
produced or expressed using the media and/or feed compositions of
the invention include, but are not limited to, extracellular
proteins, like laminins, fibronectin, integrins, etc., enzymes like
caspases, proteases, subtilisin, kinases, RNAses., DNAses, etc.,
peptide hormones like insulin, PTHrP, etc., intracellular proteins
including membrane proteins, receptors, nuclear proteins,
endoplasmic reticular proteins, etc., antibodies, any antibody
fragments like heavy- or light chains of the antibody, an antigen
binding site or motif, a chimeric antibody etc. A chimeric antibody
may be a species/species chimera or a class/class chimera. The
expressed protein or polypeptide that may be produced using the
compositions and media/feeds of the invention may be a human or
mammalian derived protein sequence that is expressed in a
non-animal cell line, like a plant cell, in order to produce animal
origin-free proteins that are free of adventitious agents for
downstream use in therapeutics.
[0145] Methods of producing a polypeptide according to the
invention comprise cultivating a cell (e.g., a mammalian cell) that
has been genetically engineered to produce a polypeptide in the
small peptide- or dipeptide-containing cell culture media described
herein under conditions suitable for expression of the polypeptide
by the cell. Optimal methods for genetically engineering a
mammalian cell to express a polypeptide of interest are well-known
in the art and will therefore be familiar to one of ordinary skill.
See e.g., Cell Culture Technology for Pharmaceutical and Cell-Based
Therapies, 15-40 (Sadettin Ozturk and Wei-Shou Hu eds., Taylor and
Francis Group 2006). Cells may be genetically engineered prior to
cultivation in the media of the invention, or they may be
transfected with one or more exogenous nucleic acid molecules after
being placed into culture in the media. Genetically engineered
cells may be cultivated in the present culture media either as
monolayer cultures, or more preferably as suspension cultures
according to the methods described above. Following cultivation of
the cells, the polypeptide of interest may optionally be purified
from the cells and/or the used culture medium according to
techniques of protein isolation that will be familiar to one of
ordinary skill in the art.
[0146] 10. Detecting Small Peptides in Culture Media or in
Concentrated Feeds
[0147] The small peptides and dipeptides described herein can be
detected using any technique known in the art for detecting amino
acids and/or small peptides, including but not limited to, acid
hydrolysis, liquid chromatography, capillary electrophoresis (Brown
et al., J. Chrom. (1994) A, 661: 279-285), HPLC (van Wandelen et
al., J. Chrom. (1997) A, 763: 11-22), or mass spectrometry. Acid
hydrolysis of peptides for chromatographic analysis of amino acid
compositions and concentration is well-known in the art. Comparison
of the chromatographic profiles of the amino acid peaks before and
after acid hydrolysis can indicate the composition and
concentration of amino acids, and therefore small peptides in a
media, feed or supplement. For instance, if tyrosine were present
in a small peptide in a media, its concentration would increase in
the acid hydrolysate of the media sample (for e.g., the tyrosine
peak height and/or area of the peak) compared to the peak
height/area of the identical media sample before acid
hydrolysis.
[0148] By way of example, the alanyl tyrosine and alanyl cysteine
dipeptides described herein were detected using the methodology
described in van Wandelen et al., J. Chrom. (1997) A, 763: 11-22,
which involved the HPLC (High Performance Liquid Chromatograph)
separation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
(AQC)--derivatized amino acid mixtures on an AccQ-Tag Ultra.TM.
(Waters Corp., Milford, Mass.) column (2.1.times.100 mm, 1.7
.mu.m). The detector parameters were set as follows: wavelength
mode: single wavelength; wavelength: 260 nm; sampling rate: 20
(points/sec); time constant: 0.4000 (sec). Using these parameters,
in an exemplary run, a sample of the alanyl tyrosine dipeptide had
a peak elution time of about 6.172 minutes (see FIG. 2),
immediately after the internal standard (AABA), and in another
exemplary run, a sample of the alanyl cysteine dipeptide had a peak
elution rate of 6.631 minutes, between lysine and tyrosine (see
FIG. 3).
[0149] This AccQ-Tag.TM. Ultra (Waters Corp., Milford, Mass.)
Pre-Column Derivatization with Ultra High Performance Liquid
Chromatograph (UPLC) method, thus, provides one way to test any
sample media or feed supplement to see if it contains a small
peptide comprising cysteine or tyrosine, for instance the
dipeptides, alanyl tyrosine or alanyl cysteine. The same method
could also be used to detect other small peptides, including other
dipeptides of interest.
[0150] In addition, the dipeptides described herein can also be
quantified with liquid chromatography with mass spectrometry
detection (LC/MS). Separation was performed with reverse phase
liquid chromatography column (Acquity UPLC.RTM. HSS T3 1.8-.mu.m,
2.1-mm i.d.times.150-mm at 40.degree. C.) coupled to a quadruple
time-of-flight mass spectrometer (Waters.RTM. SYNAPT.TM. HDMS.TM.
System, Milford, Mass.). Perfluorinated carboxylic acid (for e.g.,
perfluoropentanoic acid) was used as the ion pairing agent for
optimum separation of polar compounds (Jun Qu, Yiming Wang, Guan
Luo, Zhuping Wu, and Chengdui Yang, Anal. Chem., 2002, 74,
2034-2040; hereby incorporated by reference in its entirety).
Mobile phase-A was 0.1% formic acid and 0.05% perfluoropentanoic
acid in water, and mobile phase-B was 0.1% formic acid and 0.05%
perfluoropentanoic acid in 80% acetonitrile. All reagents were
LC/MS grade. Ultra-performance liquid chromatography (UPLC)
gradient was linear over 1-45% of mobile phase-B with a 0.4-mL/min
flow rate for 15 min. Mass spectra was collected every 0.5-sec
within 70-1000 Da in continuum, positive electrospray (+ES), and
V-mode. Protonated L-alanyl-L-cysteine dimer and protonated
L-alanyl-L-tyrosine ions were extracted from total ion
chromatograms as 383.105 (.+-.0.03) Da and 253.118 (.+-.0.03) Da,
respectively. Retention times for protonated L-alanyl-L-cysteine
dimer and protonated L-alanyl-L-tyrosine ions were 9.60 (.+-.0.03)
min and 8.75 (.+-.0.03) min, respectively (FIGS. 4 to 7).
[0151] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. In case of conflict, the present
specification, including definitions, will control. 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. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their
entirety.
EXAMPLE 1
Production of Dipeptide-Containing Culture Medium
[0152] An exemplary cell culture medium containing the dipeptides
alanyl tyrosine (AlaTyr) and alanyl cysteine (which forms a
disulfide dimer, [AlaCys].sub.2) was prepared. Specifically, the
dipeptides, alanyl tyrosine and alanyl cysteine were added as dry
powder to an aqueous basal cell culture medium containing glucose
and a mixture of concentrated amino acids and mixed until
dissolved. Alanyl tyrosine was added to the cell culture medium at
a concentration of about 4.0 g/L. Alanyl cysteine was added to the
cell culture medium at a concentration of about 3.0 g/L. The
observed solubilities of the dipeptides in water were determined to
be about 15.4 g/L for AlaTyr, >100 g/L for AlaCys, and about 8.7
g/L for [AlaCys].sub.2 dimer. By way of comparison, the solubility
of L-Tyrosine in water is about 0.38 g/L at 20.degree. C. Free
L-Cysteine is easily oxidized into cystine. L-Cysteine
hydrochloride is considerably more stable in an acidic aqueous
solution, but in neutral or alkaline aqueous solutions, it is also
converted to L-Cystine by aerobic oxidation. The solubility of
L-Cystine in water was about 0.11 g/L at 25.degree. C.
EXAMPLE 2
Fed Batch Testing
[0153] Integrated fed batch testing was performed using
IgG-producing CHO cells grown in a basal cell culture medium CD
FortiCHO.TM. (Invitrogen Cat. No. A-1148301 and Custom Stock
A-11437DK; Life Technologies Corp., Carlsbad, Calif.) and
supplemented with either glucose (CD FortiCHO.TM.+Glucose; FIG. 1:
curve with filled squares () and forward slash-filled bar () or,
with a mixture of concentrated amino acid mixture with low levels
of cysteine or tyrosine, with glucose+the dipeptide (DP) containing
culture medium or feed, described in Example 1 (CD
FortiCHO.TM.+aa+DP Feed FP2 and FP3; see FIG. 1: (FP2) curve with
filled circles () and unfilled bar (.quadrature.); (FP3) curve with
filled diamonds () and backward slash-filled bar (). "aa" in FIG. 1
refers to a mixture of concentrated amino acid mixture with low
levels of cysteine and tyrosine, with glucose.
[0154] Other basal cell culture media studied were either CD
OptiCHO.TM., CD CHO; all from Life Technologies Corp., Carlsbad,
Calif. (data not shown). The preferred medium used was CD
FortiCHO.TM.. Exemplary mixtures of concentrated amino acids are,
for instance, CHO CD EfficientFeed.TM. A (Invitrogen Cat. No.
A1023401), CHO CD EfficientFeed.TM. B (Invitrogen Cat. No.
A1024001), CHO CD Efficient Feed.TM. kit (Invitrogen Cat. No.
A1024101), CD Efficient Feed.TM. C AGT.TM. (Custom Stock A-11525SA,
Life Technologies Corp., Carlsbad, Calif.). The cysteine and
tyrosine containing small peptides in the cell culture media or
supplemental feeds of the invention are designed to be used with
any exemplary basal cell culture medium, with any exemplary,
stoichiometrically-balanced, mixture of concentrated amino acids
(aa) optionally with glucose, vitamins, trace elements, etc.,
suitable for the growth of a desired cell type, as can be deemed by
one skilled in the art.
[0155] The cells were grown in DasGip bioreactors with 500 mL
working volume, with a pH control set point of 7.0+/-0.05 and a pO2
control set point of 30%. CHO cells supplemented with glucose were
automatically fed 3 g/L of glucose whenever the glucose level
reached 2 g/L. CHO cells supplemented with dipeptides (DP) were
subjected to two feeding schedules. In the first feeding schedule
(see FIG. 1, red curve and bar: FP2), CHO cells were fed 2% of the
dipeptide-containing medium daily from day 4 to day 13. In the
second schedule, (see FIG. 1, green curve and bar: FP3), CHO cells
were fed 2% of the dipeptide-containing medium daily from day 5 to
day 14.
[0156] Cells grown in CD FortiCHO.TM. medium and supplemented with
glucose had comparable or better IgG productivity (1600 mg/L) than
CD Opti CHO.TM. (Life Technologies, Corp., Carlsbad, Calif.) or CD
CHO (Life Technologies, Corp., Carlsbad, Calif. Carlsbad, Calif.)
based fed-batch processes (data not shown). CHO cells grown in CD
FortiCHO.TM. and supplemented with the concentrated amino acids and
the dipeptide-containing medium of Example 1 had improved viability
after 12 days and doubled productivity levels, with an IgG titer of
about 3200 mg/L by day 15, as compared to an IgG titer of about
1600 mg/L for CD FortiCHO.TM.+Glucose at day 15 (FIG. 1). Of the
two feeding schedules for CD FortiCHO.TM.+aa+Dipeptide (DP) Feed,
both worked well and showed comparable levels of IgG production.
The FP3 profile had a better maintained glucose level and a more
constant level of IgG production than FP2, while the FP2 profile
promoted a higher peak cell density. Notably, the
dipeptide-containing cell culture medium yielded enhanced cell
density and productivity levels at a significantly reduced
volumetric feed proportion, providing yet another advantage of
using the dipeptide-containing cell culture medium described
herein.
[0157] A liquid cell culture medium or feed solution containing the
dipeptides alanyl tyrosine and alanyl cysteine can be stored at
2-8.degree. C., has remained precipitate free for over 10 months,
thus demonstrating significantly longer liquid stability than
expected for such a concentrated solution.
[0158] All patents, patent applications, and published references
cited herein are hereby incorporated by reference in their
entirety. While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *