U.S. patent application number 14/016035 was filed with the patent office on 2014-01-02 for secretory protein biomarkers for high efficiency protein expression.
This patent application is currently assigned to ABBVIE INC.. The applicant listed for this patent is ABBVIE INC.. Invention is credited to Ivan Correia, Patrick Hossler.
Application Number | 20140004531 14/016035 |
Document ID | / |
Family ID | 43431809 |
Filed Date | 2014-01-02 |
United States Patent
Application |
20140004531 |
Kind Code |
A1 |
Hossler; Patrick ; et
al. |
January 2, 2014 |
Secretory Protein Biomarkers For High Efficiency Protein
Expression
Abstract
The instant invention relates to the field of protein
production, and in particular is relates to compositions and
processes for improving the production levels of recombinant
proteins expressed in host cells.
Inventors: |
Hossler; Patrick;
(Worcester, MA) ; Correia; Ivan; (Winchester,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ABBVIE INC. |
North Chicago |
IL |
US |
|
|
Assignee: |
ABBVIE INC.
North Chicago
IL
|
Family ID: |
43431809 |
Appl. No.: |
14/016035 |
Filed: |
August 30, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12941347 |
Nov 8, 2010 |
8524458 |
|
|
14016035 |
|
|
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|
61259527 |
Nov 9, 2009 |
|
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Current U.S.
Class: |
435/7.4 ; 435/15;
435/18; 435/24; 435/26; 435/29; 436/501 |
Current CPC
Class: |
G01N 33/56966 20130101;
G01N 33/5005 20130101; G01N 33/5023 20130101; G01N 33/68
20130101 |
Class at
Publication: |
435/7.4 ; 435/29;
436/501; 435/15; 435/26; 435/18; 435/24 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1. A method of identifying a high efficiency cell or cell culture
comprising: (a) determining the expression level of a high
efficiency cell or cell culture biomarker panel in the cell or cell
culture, wherein said biomarker panel comprises: Heat Shock Cognate
71 kDa Protein, Major Vault Protein, and Eukaryotic Translation
Initiation Factor 5A-1, and (b) comparing the expression level of
that high efficiency cell or cell culture biomarker panel in the
cell or cell culture to the expression level of the biomarker panel
in a known high efficiency cell or cell culture, wherein an
expression level of Heat Shock Cognate 71 kDa Protein, Major Vault
Protein, and Eukaryotic Translation Initiation Factor 5A-1 lower
than the level in the known high efficiency cell or cell culture is
indicative of a high efficiency cell or cell culture.
2. The method of claim 1 wherein the biomarker panel comprises an
additional secretory protein.
3. The method of claim 2 wherein the additional secretory protein
is an endoplasmic reticulum fraction-resident protein.
4. The method of claim 2 wherein the additional secretory protein
is a Golgi fraction-resident protein.
5. The method of claim 2 wherein the additional secretory protein
is up-regulated in a high efficiency cell or cell culture.
6. The method of claim 2 wherein the additional secretory protein
is down-regulated in a high efficiency cell or cell culture.
7. The method of claim 2 wherein the level of multiple additional
secretory proteins are determined and compared.
8. The method of claim 7 wherein all of the additional secretory
proteins are down-regulated in the high efficiency cell or cell
culture.
9. The method of claim 7 wherein all of the additional secretory
proteins are up-regulated in the high efficiency cell or cell
culture.
10. The method of claim 7 wherein one or more of the additional
secretory proteins is up-regulated while one or more of the
additional secretory proteins is down-regulated in the high
efficiency cell or cell culture.
11. The method of claim 2 wherein the additional secretory protein
is selected from the group consisting of: Endoplasmin precursor
(GRP 94); Seryl-tRNA synthetase; Dihydrolipolylysine residue
acetyltransferase/methionineaminopeptidase 2; F-actin capping
protein subunit alpha 2; 60s acidic ribosomal protein PO;
Heterogeneous nuclear ribonucleoprotein K; Eukaryotic translation
initiation factor 3 subunit 7; Endoplasmin precursor (GRP 94);
Elongation factor 1 beta; Seryl-tRNA synthetase/RAS
GTPase-activating protein binding 2; Eukaryotic translation factor
3 subunit 3; Pre mRNA processing factor 19/T-complex protein 1
subunit beta; Elongation factor 1 gamma; Tubulin gamma-1
chain/Eukaryotic translation initiation factor 2 subunit 2;
Vimentin; Myosin regulatory light chain; Guanine nucleotide binding
protein subunit beta 1; 26S Protease regulatory subunit 6B;
Eukaryotic peptide chain releases factor subunit 1; Protein
disulfide isomerase A-3 precursor; Tubulin alpha-2; Endoplasmin
precursor (GRP 94); Protein disulfide isomerase A-6
precursor/Protein NDRG1 (N-myc downstream-regulated gene 1
protein); 60 kDa heat shock protein, mitochondrial precursor
(Hsp60); T-complex protein 1 subunit epsilon; UPF0027 protein; 26S
Proteasome non ATP-ase regulatory subunit 13; SH3 domain GRB2-like
protein B1; COP9 sinalosome complex subunit 4; Cytoplasmic dynein 1
intermediate chain; Ribosome Binding Protein 1; Alcohol
Dehydrogenase (NADP+); Glutamate dehydrogenase 1 (mitochondrial
precursor); Proteasome activator complex subunit 1; Cytoplasmic 2,
Actin; Annexin A5; Coatomer subunit Epsilon; Glucosidase 2;
Triosephosphate isomerase; Heterogeneous nuclear ribonucleoprotein
F; Cytoplasmic 2/Actin; Sorting Nexin 6/Synaptic vesicle membrane
VAT-1 homolog; 14-3-3 protein zeta/delta; Vimentin/Tubulin alpha-2
chain; Vaculoar ATP Synthase; Ig-G gamma-1 chain C; EH-Domain
Containing Protein 4; Heat Shock Protein Beta 1; Translation
Initiation Factor 3 Subunit 3; ATP Synthase Subunit Beta; 60S
Acidic Ribosomal Protein PO; Ezrin; Nucleophosmin; Calreticulin;
14-3-3 Protein Gamma; Septin-11; Annexin A2; ADP-Ribosylation
Factor-Like Protein 2R; and Myosin Light Polypeptide 6.
12. A kit for identifying modulation in expression of a secretory
protein biomarker of high efficiency protein expression comprising:
(a) multiple detectable antibodies and wherein each antibody
specifically binds to a secretory protein of a biomarker panel of
high efficiency protein expression, wherein the biomarker panel
comprises Heat Shock Cognate 71 kDa Protein, Major Vault Protein,
and Eukaryotic Translation Initiation Factor 5A-1 and (b) a means
for detecting said antibody.
13. The kit of claim 12 comprising an additional detectable
antibody directed against an additional secretory protein, wherein
the additional secretory protein is selected from the group
consisting of: Endoplasmin precursor (GRP 94); Seryl-tRNA
synthetase; Dihydrolipolylysine residue
acetyltransferase/methionineaminopeptidase 2; F-actin capping
protein subunit alpha 2; 60s acidic ribosomal protein PO;
Heterogeneous nuclear ribonucleoprotein K; Eukaryotic translation
initiation factor 3 subunit 7; Endoplasmin precursor (GRP 94);
Elongation factor 1 beta; Seryl-tRNA synthetase/RAS
GTPase-activating protein binding 2; Eukaryotic translation factor
3 subunit 3; Pre mRNA processing factor 19/T-complex protein 1
subunit beta; Elongation factor 1 gamma; Tubulin gamma-1
chain/Eukaryotic translation initiation factor 2 subunit 2;
Vimentin; Myosin regulatory light chain; Guanine nucleotide binding
protein subunit beta 1; 26S Protease regulatory subunit 6B;
Eukaryotic peptide chain releases factor subunit 1; Protein
disulfide isomerase A-3 precursor; Tubulin alpha-2; Endoplasmin
precursor (GRP 94); Protein disulfide isomerase A-6
precursor/Protein NDRG1 (N-myc downstream-regulated gene 1
protein); 60 kDa heat shock protein, mitochondrial precursor
(Hsp60); T-complex protein 1 subunit epsilon; UPF0027 protein; 26S
Proteasome non ATP-ase regulatory subunit 13; SH3 domain GRB2-like
protein B1; COP9 sinalosome complex subunit 4; Cytoplasmic dynein 1
intermediate chain; Ribosome Binding Protein 1; Alcohol
Dehydrogenase (NADP+); Glutamate dehydrogenase 1 (mitochondrial
precursor); Proteasome activator complex subunit 1; Cytoplasmic 2,
Actin; Annexin A5; Coatomer subunit Epsilon; Glucosidase 2;
Triosephosphate isomerase; Heterogeneous nuclear ribonucleoprotein
F; Cytoplasmic 2/Actin; Sorting Nexin 6/Synaptic vesicle membrane
VAT-1 homolog; 14-3-3 protein zeta/delta; Vimentin/Tubulin alpha-2
chain; Vaculoar ATP Synthase; Ig-G gamma-1 chain C; EH-Domain
Containing Protein 4; Heat Shock Protein Beta 1; Translation
Initiation Factor 3 Subunit 3; ATP Synthase Subunit Beta; 60S
Acidic Ribosomal Protein PO; Ezrin; Nucleophosmin; Calreticulin;
14-3-3 Protein Gamma; Septin-11; Annexin A2; ADP-Ribosylation
Factor-Like Protein 2R; and Myosin Light Polypeptide 6.
14. The kit of claim 13 wherein the kit comprises multiple
additional detectable antibodies and wherein each antibody
specifically binds to a different additional secretory protein
selected from the group consisting of Endoplasmin precursor (GRP
94); Seryl-tRNA synthetase; Dihydrolipolylysine residue
acetyltransferase/methionineaminopeptidase 2; F-actin capping
protein subunit alpha 2; 60s acidic ribosomal protein PO;
Heterogeneous nuclear ribonucleoprotein K; Eukaryotic translation
initiation factor 3 subunit 7; Endoplasmin precursor (GRP 94);
Elongation factor 1 beta; Seryl-tRNA synthetase/RAS
GTPase-activating protein binding 2; Eukaryotic translation factor
3 subunit 3; Pre mRNA processing factor 19/T-complex protein 1
subunit beta; Elongation factor 1 gamma; Tubulin gamma-1
chain/Eukaryotic translation initiation factor 2 subunit 2;
Vimentin; Myosin regulatory light chain; Guanine nucleotide binding
protein subunit beta 1; 26S Protease regulatory subunit 6B;
Eukaryotic peptide chain releases factor subunit 1; Protein
disulfide isomerase A-3 precursor; Tubulin alpha-2; Endoplasmin
precursor (GRP 94); Protein disulfide isomerase A-6
precursor/Protein NDRG1 (N-myc downstream-regulated gene 1
protein); 60 kDa heat shock protein, mitochondrial precursor
(Hsp60); T-complex protein 1 subunit epsilon; UPF0027 protein; 26S
Proteasome non ATP-ase regulatory subunit 13; SH3 domain GRB2-like
protein B1; COP9 sinalosome complex subunit 4; Cytoplasmic dynein 1
intermediate chain; Ribosome Binding Protein 1; Alcohol
Dehydrogenase (NADP+); Glutamate dehydrogenase 1 (mitochondrial
precursor); Proteasome activator complex subunit 1; Cytoplasmic 2,
Actin; Annexin A5; Coatomer subunit Epsilon; Glucosidase 2;
Triosephosphate isomerase; Heterogeneous nuclear ribonucleoprotein
F; Cytoplasmic 2/Actin; Sorting Nexin 6/Synaptic vesicle membrane
VAT-1 homolog; 14-3-3 protein zeta/delta; Vimentin/Tubulin alpha-2
chain; Vaculoar ATP Synthase; Ig-G gamma-1 chain C; EH-Domain
Containing Protein 4; Heat Shock Protein Beta 1; Translation
Initiation Factor 3 Subunit 3; ATP Synthase Subunit Beta; 60S
Acidic Ribosomal Protein PO; Ezrin; Nucleophosmin; Calreticulin;
14-3-3 Protein Gamma; Septin-11; Annexin A2; ADP-Ribosylation
Factor-Like Protein 2R; and Myosin Light Polypeptide 6.
15. The kit of claim 13 wherein the kit comprises a positive
control sample.
16. The kit of claim 13 wherein the kit comprises a negative
control sample.
Description
1. CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of U.S.
application Ser. No. 12/941,347, filed Nov. 8, 2010, which claims
the benefit of U.S. Provisional Application Ser. No. 61/259,527,
filed Nov. 9, 2009, both of which are hereby incorporated by
reference in their entirety.
2. INTRODUCTION
[0002] The instant invention relates to the field of protein
production, and in particular relates to compositions and processes
for improving the production levels of proteins expressed in host
cells.
3. BACKGROUND OF THE INVENTION
[0003] The production of recombinant proteins for biopharmaceutical
applications typically involves the use of cell cultures having
variable protein expression efficiencies. Advances in technologies
for gene mapping, bio-imaging, and whole proteome analysis provide
unique opportunities to understand many of the bottlenecks
associated with the use of cell cultures for large-scale production
of recombinant proteins. Numerous presentations at recent
conferences (IBC-Antibody Development and Production, San Diego,
Mar. 12-14, 2008), as well as publications in peer-reviewed
journals (Gupta, P. and K. H. Lee (2007), "Genomics and proteomics
in process development: opportunities and challenges" Trends
Biotechnol 25(7): 324-30.) have demonstrated the utility of such
technologies in the biopharmaceutical industry. The objectives for
many of these studies include improving cell productivity,
improving cell culture survival and proliferation, as well as
introducing rapid and reliable techniques for the selection of high
producing cell lines and real-time monitoring of the viability and
physiology of the cells.
[0004] Even in light of the above-described advances, there remains
a need in the art to identify biomarkers that correlate with high
efficiency protein expression, particularly in the context of cell
culture processes used for commercially produced recombinant
bio-therapeutics. The instant invention addresses that need by
providing secretory protein biomarkers that correlate with high
efficiency protein expression
4. SUMMARY OF THE INVENTION
[0005] The present invention is directed to secretory protein
biomarkers that correlate with high efficiency protein expression
as well as methods of using such biomarkers to facilitate high
efficiency protein expression, and especially recombinant protein
expression, in host cells.
[0006] In certain embodiments the secretory protein biomarker is an
endoplasmic reticulum fraction-resident protein. In certain
embodiments the secretory protein biomarker is a Golgi
fraction-resident protein. In certain embodiments the secretory
protein biomarker is derived from a total cell fraction. In certain
embodiments the secretory protein biomarker is up-regulated in a
high efficiency cell or cell culture. In certain embodiments the
secretory protein biomarker is down-regulated in a high efficiency
cell or cell culture
[0007] In certain embodiments of the present invention, a secretory
protein biomarker is employed to identify a high efficiency cell or
cell culture. In certain embodiments multiple secretory protein
biomarkers are employed to identify a high efficiency cell or cell
culture. In certain embodiments the up-regulation of a secretory
protein biomarker is employed to identify a high efficiency cell or
cell culture. In certain embodiments the down-regulation of a
secretory protein biomarker is employed to identify a high
efficiency cell or cell culture. In certain embodiments where
multiple secretory protein biomarkers are employed to identify a
high efficiency cell or cell culture, all of secretory protein
biomarkers are up-regulated. In other embodiments involving
multiple secretory protein biomarkers, all of the secretory protein
biomarkers are down-regulated. In yet further embodiments involving
multiple secretory protein biomarkers, one or more of the secretory
protein biomarkers is up-regulated while one or more of the
secretory protein biomarkers is down-regulated.
5. BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1 depicts the effect of different cell culture
treatments on product titers in development bioreactors.
(IVC=integral of viable cell density, Qp=specific
productivity).
[0009] FIG. 2 depicts Transmission Electron Microscopic (TEM)
images of purified ER and Golgi fractions.
[0010] FIG. 3 depicts 2D-gel electrophoresis of total cell lysates
versus fractionated ER and Golgi samples.
[0011] FIG. 4 depicts a mass spectrometry workflow diagram for
identification of differentially expressed protein spots excised
from 2D gels.
[0012] FIG. 5 depicts the relative value (Culture 1/Culture 2) of
the product titer, specific productivity (q.sub.p), and integral of
viable cell density (IVC) obtained during the culture processes of
Cell Line 1 described in Section 7.2.2.
[0013] FIG. 6 depicts the relative expression (x-axis) versus
number of proteins (y-axis) falling either less than -0.4 or
greater than 0.4 in endoplasmic reticulum enriched fraction samples
taken at days 5, 8, and 11 from the culture processes of Cell Line
1 described in Section 7.2.2.
[0014] FIG. 7 depicts the relative expression (x-axis) versus
number of proteins (y-axis) falling either less than -0.4 or
greater than 0.4 in golgi enriched fraction samples taken at days
5, 8, and 11 from the culture processes of Cell Line 1 described in
Section 7.2.2.
[0015] FIG. 8 depicts the relative value (Culture 1/Culture 2) of
the product titer, specific productivity (q.sub.p), and integral of
viable cell density (IVC) obtained during the culture process of
Cell Line 2 described in Section 7.2.3.
[0016] FIG. 9 depicts the relative expression (x-axis) versus
number of proteins (y-axis) falling either less than -0.4 or
greater than 0.4 in endoplasmic reticulum enriched fraction samples
taken at days 8 and 11 from the culture processes of Cell Line 2
described in Section 7.2.3.
[0017] FIG. 10 depicts the relative expression (x-axis) versus
number of proteins (y-axis) falling either less than -0.4 or
greater than 0.4 in golgi enriched fraction samples taken at days
5, 8, and 11 from the culture processes of Cell Line 2 described in
Section 7.2.3.
[0018] FIG. 11 depicts the relative value (Culture 1/Culture 2) of
the product titer, specific productivity (q.sub.p), and integral of
viable cell density (IVC) obtained during the culture process of
Cell Line 3 described in Section 7.2.4.
[0019] FIG. 12 depicts the relative expression (x-axis) versus
number of proteins (y-axis) falling either less than -0.4 or
greater than 0.4 in endoplasmic reticulum enriched fraction samples
taken at days 5/6, 8/9, and 11/13 from the culture processes of
Cell Line 3 described in Section 7.2.4.
[0020] FIG. 13 depicts the relative expression (x-axis) versus
number of proteins (y-axis) falling either less than -0.4 or
greater than 0.4 in golgi reticulum enriched fraction samples taken
at days 5/6, 8/9, and 11/13 from the culture processes of Cell Line
3 described in Section 7.2.4.
6. DETAILED DESCRIPTION OF THE INVENTION
5.1 Definitions
[0021] As used herein, the term "high efficiency protein
expression" refers to a phenotype of a cell or cell culture wherein
the cell or cell culture expresses a protein of interest at a
higher concentration than other comparable cells. This phenotype
can be observed under general cell culture conditions or may
require the use of particular cell culture conditions to elicit the
phenotype. A cell manifesting high efficiency protein expression is
referred to herein as a "high efficiency cell" and a culture of
said cells is a "high efficiency cell culture."
[0022] As used herein, the term "recombinant host cell" (or simply
"host cell") refers to a cell into which a recombinant expression
vector has been introduced. It should be understood that such terms
are intended to refer not only to the particular subject cell but
to the progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term "host cell" as used herein. In certain
embodiments the host cell is employed in the context of a cell
culture.
[0023] As used herein, the term "cell culture" refers to methods
and techniques employed to generate and maintain a population of
host cells capable of producing a recombinant protein of interest,
as well as the methods and techniques for optimizing the production
and collection of the protein of interest. For example, once an
expression vector has been incorporated into an appropriate host,
the host can be maintained under conditions suitable for high level
expression of the relevant nucleotide coding sequences, and the
collection and purification of the desired recombinant protein.
Mammalian cells are preferred for expression and production of the
recombinant of the present invention, however other eukaryotic cell
types can also be employed in the context of the instant invention.
See, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y.,
N.Y. (1987). Suitable mammalian host cells for expressing
recombinant proteins according to the invention include Chinese
Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in
Urlaub and Chasin, (1980) PNAS USA 77:4216-4220, used with a DHFR
selectable marker, e.g., as described in Kaufman and Sharp (1982)
Mol. Biol. 159:601-621, the entire teachings of which are
incorporated herein by reference), NS0 myeloma cells, COS cells and
SP2 cells. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);
human embryonic kidney line (293 or 293 cells subcloned for growth
in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977));
baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary
cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216
(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251
(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green
monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical
carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC
CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human
lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB
8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells
(Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5
cells; FS4 cells; and a human hepatoma line (Hep G2), the entire
teachings of which are incorporated herein by reference.
[0024] As used herein a "recombinant expression vector" can be any
suitable recombinant expression vector, and can be used to
transform or transfect any suitable host. For example, one of
ordinary skill in the art would appreciate that transformation or
transfection is a process by which exogenous nucleic acid such as
DNA is introduced into a cell wherein the transformation or
transfection process involves contacting the cell with the
exogenous nucleic acid such as the recombinant expression vector as
described herein. Non-limiting examples of such expression vectors
are the pUC series of vectors (Fermentas Life Sciences), the
pBluescript series of vectors (Stratagene, LaJolla, Calif.), the
pET series of vectors (Novagen, Madison, Wis.), the pGEX series of
vectors (Pharmacia Biotech, Uppsala, Sweden), and the pEX series
vectors (Clontech, Palo Alto, Calif.).
[0025] As used herein, the term "recombinant protein" refers to a
protein produced as the result of the transcription and translation
of a gene carried on a recombinant expression vector that has been
introduced into a host cell. In certain embodiments the recombinant
protein is an antibody, preferably a chimeric, humanized, or fully
human antibody. In certain embodiments the recombinant protein is
an antibody of an isotype selected from group consisting of: IgG
(e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In
certain embodiments the antibody molecule is a full-length antibody
(e.g., an IgG1 or IgG4 immunoglobulin) or alternatively the
antibody can be a fragment (e.g., a Fc fragment or a Fab
fragment).
[0026] As used herein, the term "secretory protein" refers to a
protein resident, even transiently, in the secretory apparatus of a
eukaryotic cell. The secretory apparatus of eukaryotic cells is
composed of the endoplasmic reticulum (ER), the ER-Golgi
intermediate compartment (ERGIC) Golgi apparatus (Ladinsky, et al.
(1999), "Golgi structure in three dimensions: functional insights
from the normal rat kidney cell" J Cell Biol 144(6): 1135-49), in
addition to the vesicles involved in transport between them, as
well as protein degradation mechanisms via the proteasome and
lysosome. Electron microscopy and fractionation techniques have
also been used to describe the major organelles of the secretory
pathway (Sabatini, D. D. (1999), "George E. Palade: charting the
secretory pathway" Trends Cell Biol 9(10): 413-7). A large number
of proteins and other effector molecules are involved in secretory
transport. Recently, researchers at McGill University have
documented the presence of over 1400 proteins in the secretory
pathway proteome (Gilchrist, A., C. E. Au, et al. (2006),
"Quantitative proteomics analysis of the secretory pathway" Cell
127(6): 1265-81). The authors further demonstrated exquisite
spatial information of the proteins in different compartments of
the cell. Of the 1400 proteins identified, over 300 of them are of
unknown function.
[0027] As used herein the term "endoplasmic reticulum" or "ER"
refers to a eukaryotic cell organelle that forms an interconnected
network of tubules, vesicles, and cisternae within cells that is
involved in the production of phospholipids and proteins, among
other functions. The ER facilitates proper protein folding and
quality control (QC) for protein processing. During translation,
proteins are translocated into the ER lumen after the signal
recognition particle helps dock the translation complex to its
receptor on the ER membrane. The nascent polypeptide is transferred
to the translocon where it is subsequently passed into the lumen of
the ER during peptide elongation, for proper folding and
glycosylation within the highly oxidizing microenvironment.
Cellular control mechanisms help prevent the transport of unfolded,
misfolded, or unassembled proteins out of the ER. Protein folding
and glycosylation initiation immediately take place in the ER after
the polypeptide chain is translocated into the ER lumen. Through
the concerted action of these proteins and others, a network of
interactions occurs, and the molecular cargo is processed and
screened. Numerous chaperone proteins facilitate protein folding,
including heat shock proteins which temporarily block
intermolecular interactions during folding, protein disulfide
isomerase (PDI) which catalyzes the formation of correct disulfide
bonds, and calnexin and calreticulin which serve as a quality
control step towards ensuring that proteins are properly folded
before leaving the ER (Hossler, P. (2006), "Experimental and
Theoretical Exploration of Protein Glycosylation in Mammalian Cell
Culture" Ph.D. Thesis ProQuest/UMI). Chaperone proteins are major
ER-resident proteins for ensuring the proper folding structure of
recombinant proteins. The most abundant ER chaperone is GRP78/BiP,
which uses ATP hydrolysis to promote protein folding, and to
prevent aggregation of unfolded proteins within the ER.
[0028] As used herein the term "Golgi apparatus" or "Golgi" refers
to a compound membranous cytoplasmic organelle of eukaryotic cells,
consisting of flattened, ribosome-free vesicles arranged in a more
or less regular stack. Once proteins enter the Golgi apparatus,
they are processed by a myriad of protein glycosylation enzymes. It
is in the Golgi that the N- and O-glycans are further extended,
further increasing the diversity of the glycoform profile. The
first reactions for N-glycan processing are further removal of
mannose sugars that are remaining from ER processing. After
removal, a small number of glycosyltransferase enzymes react upon
the glycans, but in variable order, leading to a diverse array of
product glycans. This network of reactions, as well as the
enzymatic substrate specificities has been documented previously
(Hossler, P., L. T. Goh, et al. (2006), "GlycoVis: visualizing
glycan distribution in the protein N-glycosylation pathway in
mammalian cells" Biotechnol Bioeng 95(5): 946-60). This final
glycoform profile has been shown to be affected by the nature of
this network, as well as numerous other factors including the
localization of the enzymes across the Golgi cisternae, relative
nucleotide-sugar concentrations, and the protein residence time
within the organelle.
[0029] As used herein, the term "up-regulated" refers to an
increase in the amount of a particular composition, such as, but
not limited to, a protein. Such increases in protein amount can be
the result of, for example, but not by way of limitation: changes
in transcription or translation rate of the DNA or mRNA,
respectively, that encode the protein; changes in the stability of
the mRNA that encodes the protein; and/or changes in the stability
of the protein itself. In certain embodiments the increase in
amount can be increases of about 1%, about 10%, about 25%, about
50%, about 100% or greater. In preferred embodiments, the increase
in amount is an increase of at least about 100% to about
1,000%.
[0030] As used herein, the term "down-regulated" refers to an
decrease in the amount of a particular composition, such as, but
not limited to, a protein. Such decreases in protein amount can be
the result of, for example, but not by way of limitation: changes
in transcription or translation rate of the DNA or mRNA,
respectively, that encode the protein; changes in the stability of
the mRNA that encodes the protein; and/or changes in the stability
of the protein itself. In certain embodiments the decrease in
amount can be decreases of about 1%, about 10%, about 25%, about
50%, about 100% or greater. In preferred embodiments, the decrease
in amount is a decrease of at least about 100% to about 1,000%.
[0031] The term "about", as used herein, is intended to refer to
ranges of approximately 10-20% greater than or less than the
referenced value. In certain circumstances, one of skill in the art
will recognize that, due to the nature of the referenced value, the
term "about" can mean more or less than a 10-20% deviation from
that value.
6.2 Biomarkers
[0032] The present invention is directed to secretory protein
biomarkers that correlate with high efficiency protein expression
as well as methods of using such biomarkers to facilitate high
efficiency protein expression, and especially recombinant protein
expression, in cells. Such biomarkers can be identified using a
variety of techniques. For example, but not by way of limitation,
proteomic analysis of a high efficiency cell cultures can be
employed to identify secretory proteins exhibiting differential
expression in high efficiency cell cultures as compared to lower
efficiency cell cultures.
[0033] In order to identify a specific high efficiency cell or cell
culture for use in the context of the instant invention, a cell or
cells producing a protein of interest, for example a host cell or
cells producing a recombinant protein of interest, can be cultured
in a variety of different media to identify the media that elicits
a high efficiency phenotype in the resulting cell culture (which
comprises the original cell or cells and/or its/their progeny).
Commercially available media such as Ham's F10.TM. (Sigma), Minimal
Essential Medium.TM. ((MEM), (Sigma), RPMI-1640 (Sigma), and
Dulbecco's Modified Eagle's Medium.TM. ((DMEM), Sigma) are suitable
for culturing the cells. In addition, any of the media described in
Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem.
102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;
4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No.
Re. 30,985, the entire teachings of which are incorporated herein
by reference, can be used as culture media for the cells. Any of
these media types can be supplemented as necessary with hormones
and/or other growth factors (such as, but not limited to, insulin,
transferrin, or epidermal growth factor), salts (such as, but not
limited to, sodium chloride, calcium, magnesium, and phosphate),
buffers (such as, but not limited to, HEPES), nucleotides (such as,
but not limited to, adenosine and thymidine), antibiotics (such as,
but not limited to, gentamycin), trace elements (defined as
inorganic compounds usually present at final concentrations in the
micromolar range), and glucose or any equivalent energy source. Any
other necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art.
[0034] In addition to variation of media type, other culture
conditions, such as temperature, pH, and the like, can be modulated
in an effort to elicit the high expression cell culture phenotype
for use in the context of the instant invention. The type and
extent of any particular change in such conditions that will be
relevant for any one specific host cell will depend on the
particular host cell selected for expression, and will be apparent
to the ordinarily skilled artisan.
[0035] When using the cell culture techniques of the instant
invention, the protein of interest can be produced intracellularly,
in the periplasmic space, or directly secreted into the medium. In
embodiments where the protein of interest is produced
intracellularly, the particulate debris, either host cells or lysed
cells (e.g., resulting from homogenization), can be removed by a
variety of means, including but not limited to, by centrifugation
or ultrafiltration. Where the protein of interest is secreted into
the medium, supernatants from such expression systems can be first
concentrated using a commercially available protein concentration
filter, e.g., an Amicon.TM. or Millipore Pellicon.TM.
ultrafiltration unit. The amount of the protein of interest used in
determining whether a particular cell culture is a high efficiency
cell culture can be calculated by assaying the media directly, in
the case of secreted proteins, or by assaying media after the host
cells have been lysed and the media has been subjected to
centrifugation or ultrafiltration.
[0036] Once a high efficiency cell or cell culture has been
identified, or conditions for high efficiency protein expression
have been established, which result in a cell culture producing a
protein of interest in a highly efficient manner, proteomic
analysis can be undertaken to determine if there are specific
proteins that are differentially expressed in the high efficiency
cell culture as compared to a lower efficiency cell culture. The
term "proteomic analysis" is used to refer to analysis of the
expression pattern of one or more protein in a biological sample.
Such analysis can, for example, be accomplished using mass
spectrometry, two-dimensional gel electrophoresis, immunoassays, or
by any other means for quantifying the level of protein expression
in a sample.
[0037] Proteomic analysis can result in the identification of one
or more proteins that are differentially expressed in high
efficiency and low efficiency cell cultures. For example, but not
by way of limitation such analysis can identify least 2, or at
least 5 or at least 10 or at least 15, or at least 20, or at least
25, or at least 30, or at least 35, or at least 40, or at least 45,
or at least 50, or at least 60, or at least 65, or at least 70, or
at least 75, or at least 80, or at least 85, or at least 85, or at
least 90, or at least 95, or at least 100, or at least 125, or at
least 150, or at least 175, or at least 200 differentially
expressed proteins in a particular cell culture sample.
[0038] Proteomic analysis may be directed toward a particular class
of protein, for example, secretory proteins. In order to perform
proteomic analysis of candidate secretory protein biomarkers, it is
necessary to first obtain samples containing such proteins. In
certain embodiments the candidate secretory protein biomarkers are
obtained from total cell lysates. In preferred embodiments the
candidate biomarker secretory proteins are obtained from samples
enriched for secretory organelles. There are numerous
well-established protocols reported in the literature for producing
total cell lysates as well as for producing samples containing
enriched (also referred to as "purified") secretory organelles, or
subsets of such organelles. Typically, such protocols involve some
form of cell disruption via chemical or mechanical means followed
by differential, or density gradient centrifugation for
purification and subsequent assessment. Other methods include the
use of cell disruption followed by immunomagnetic separation via
antibodies towards specific organelle proteins (Vitale, N., K.
Horiba, et al. (1998), "Localization of ADP-ribosylation factor
domain protein 1 (ARD1) in lysosomes and Golgi apparatus" Proc Natl
Acad Sci USA 95(15): 8613-8). In certain embodiments, commercially
available assay kits are employed, such as the Golgi Isolation
Kit.TM. or the Endoplasmic Reticulum Isolation Kit.TM., both from
Sigma-Aldrich, which utilize buffers and/or sucrose gradient
ultracentrifugation to purify both the ER-containing and
Golgi-containing fractions. In certain embodiments the source cells
for such experiments can be obtained using small, large, or
commercial scale bioreactors, for example, but not by way of
limitation, 3 L benchtop Applikon bioreactors. In embodiments where
specific organelles are purified, verification of organelle
enrichment can be achieved using a variety of methods, including,
but not limited to visual verification by transmission electron
microscopy (TEM) morphometry.
[0039] Once a sample is prepared for proteomic analysis, the
protein composition of that sample can be identified using a
variety of means, including, but not limited to 2DGE. 2DGE is a
well-established analytical method that can resolve 1000-2000
proteins in complex mixtures by their isoelectric points in the
first dimension and then by their molecular weight using SDS-PAGE
in the second dimension. Detection of proteins that are run on 2D
gels can be accomplished by a number of different techniques,
examples of which are disclosed at proteomeconsult.com. Silver
staining is the most frequently used method for the detection of
low abundance protein spots. Silver staining has good sensitivity
(<1 ng protein/spot), however, a poor linear dynamic
quantification range (.about.one order of magnitude). In contrast,
Coomassie R-250 staining is more widely used, has a better linear
dynamic quantification range (.about.two orders of magnitude),
however, the sensitivity is low (.about.200 ng protein/spot).
Coomassie G-250 staining offers higher sensitivity (.about.25 ng
protein/spot) and is compatible with mass spectrometric procedures.
Fluorescence staining has, with the introduction of
SYPRO.RTM.-Ruby, become the choice for the 2D-gel based
quantification of proteins. Sypro.RTM. Ruby staining offers good
sensitivity (.about.1 ng protein/spot) and a linear dynamic
quantification range of .about.3 orders of magnitude. However,
fluorescence stains add a significant amount of costs to the 2D-gel
process. Finally, radio-labeling combines high sensitivity (<0.1
ng/spot) and high linear dynamic quantification range (4-5 orders
of magnitude). However, increased effort has to be applied during
gel handling, staining, scanning, storing and disposal to ensure
operator safety and to comply with environmental regulations.
[0040] Key objectives in 2-D analysis are to remove subjectivity,
to control variable gel running and gel image quality, to provide
high sensitivity for low level protein expression, and to identify
real changes in protein levels. Technology designed to meet these
challenges has been developed by Nonlinear Inc. For example,
Progenesis Discovery.TM. is a particular software solution
specifically developed for researchers in the field of proteomics
where large numbers of gels need to be accurately analyzed.
Features of this software include Intelligent Noise Correction
Algorithm (INCA), which controls the effect of image noise and
drives accurate analysis, and Data Quality Control (Data QC), where
the user receives a statistical measure of whether changes in spots
are significant compared to the overall quality of the gel. The
benefits of INCA and Data QC include high level of accuracy in spot
detection, automated approach to 2D analysis and production of
meaningful data from noisy images. In certain embodiments,
differential expression is assigned using Progenesis software,
followed by the physical excision of spots for mass spectrometry
identification. In certain embodiments, the cut-off criteria that
can be used for the assignment of differential expression is at
least about a 2-fold, about a 3-fold, or about a 5-fold increase or
decrease in normalized spot volume intensity from the 2D gels. In
alternative embodiments, the cut-off criteria that can be used for
the assignment of differential expression is a relative expression
level between two culture conditions of (positive or negative)
0.01, 0.05, 0.10, 0.2, 0.4, 0.6, 0.8, or 1.0. In certain
embodiments, relative expression can be determined using the
following equation:
Relative Expression of Protein "i"=Log.sub.10[(Normalized Spot
Volume).sub.i, culture 1/(Normalized Spot Volume).sub.i, culture
2]
[0041] Following the excision, the identity of the biomarker of
interest can be obtained using a number of techniques, including,
but not limited to, mass spectrometry. "Mass spectrometry" or "MS"
refers to an analytical technique in which a sample to be analyzed
is ionized and then introduced to produce differences based on mass
using an electric or magnetic force, and thus the masses of ions
are analyzed. There are variety of MS principles that can be used
in connection with the instant invention including, but not limited
to, ion trap MS, Fourier transform ion cyclotron resonance mass
spectrometry (FT-ICR/MS), ion scanning MS, and Q-TOF MS. In the
methods of the present invention, MS analysis can be performed
using only one technique (that is, only one mass spectrometer), or
by using a plurality of mass spectrometers that are linked to each
other, where such analysis is referred to as "MS/MS analysis".
[0042] In certain embodiments, the specific type of mass
spectrometry employed is nano-flow ESI Q-TOF MS/MS analysis.
Electrospray ionization (ESI) is a technique for transporting
biomolecules diluted in a liquid into a gaseous phase. This
desolvation method is customarily used for mass-spectrometry
identification of proteins. For example, protoeolytic enzymes are
employed to digest proteins into unique peptide segments. These
segments are then separated through reverse-phase
High-Pressure-Liquid-Chromatography (HPLC) and sequentially
electro-sprayed into a mass spectrometer. By determining the amino
acid sequence of specific peptide segments, the mass-spectrometer
yields sufficient information to identify the protein with high
confidence. The fundamental physics of the ESI process has been the
subject of numerous investigations (for reviews of recent
development in this field, see Bruins A.P., "Mechanistic aspects of
electrospray ionization," Journal of Chromatography A, vol. 794,
pp. 345-347, 1998; and Cech et al., "Practical implications of some
recent studies in electrospray ionization fundamentals," Mass
Spectrometry Reviews, vol. 20, pp. 362-387, 2001). An electrospray
produces a cloud of ions in the gaseous phase. In a nano-ESI mode
favored for applications in proteomics, the electrospray is
established by pumping an analyte solution at slow flow rates
(100-1000 nl/min) through a small bore capillary placed within a
high electric field. This specific mass spectrometry technique can
be coupled with the use of various software programs, such as, but
not limited to, Spectrum Mill software (Agilent), for
identification of the biomarker in the SwissProt database.
[0043] Specific, non-limiting examples of secretory biomarkers
modulated in a high efficiency cell or cell line are set forth in
Table 1.
TABLE-US-00001 TABLE 1 Examples of Secretory Biomarkers Endoplasmin
precursor (GRP 94) 26S Proteasome non ATP-ase regulatory subunit
13/SH3 domain GRB2-like protein B1/COP9 sinalosome complex subunit
4 Seryl-tRNA synthetase Cytoplasmic dynein 1 intermediate chain
Dihydrolipolylysine residue acetyltransferase/ Ribosome Binding
Protein 1 methionineaminopeptidase 2 F-actin capping protein
subunit alpha 2 Alcohol Dehydrogenase (NADP+) 60s acidic ribosomal
protein PO Glutamate dehydrogenase 1 (mitochondrial precursor)
Heterogeneous nuclear ribonucleoprotein K Proteasome activator
complex subunit 1 Eukaryotic translation initiation factor 3
Cytoplasmic 2, Actin subunit 7 Endoplasmin precursor (GRP 94)
Annexin A5/Coatomer subunit Epsilon Elongation factor 1 beta
Glucosidase 2 Seryl-tRNA synthetase/RAS GTPase- Triosephosphate
isomerase activating protein binding 2 Eukaryotic translation
factor 3 subunit 3 Heterogeneous nuclear ribonucleoprotein F Pre
mRNA processing factor 19/T-complex Sorting Nexin 6/Synaptic
vesicle membrane protein 1 subunit bet VAT-1 homolog Elongation
factor 1 gamma 14-3-3 protein zeta/delta Tubulin gamma-1
chain/Eukaryotic Vimentin/Tubulin alpha-2 chain translation
initiation factor 2 subunit 2 Myosin regulatory light chain
Vaculoar ATP Synthase Guanine nucleotide binding protein subunit
Ig-G gamma-1 chain C beta 1 26S Protease regulatory subunit 6B
Protein disulfide isomerase A-6 precursor/ Protein NDRG1 (N-myc
downstream- regulated gene 1 protein) Eukaryotic peptide chain
releases factor 60 kDa heat shock protein, mitochondrial subunit 1
precursor (Hsp60) Protein disulfide isomerase A-3 precursor
T-complex protein 1 subunit epsilon Tubulin alpha-2 UPF0027 protein
Endoplasmin precursor (GRP 94)
[0044] Additional exemplary biomarkers include, but are not limited
to, EH-Domain Containing Protein 4; Heat Shock Protein Beta 1;
Translation Initiation Factor 3 Subunit 3; ATP Synthase Subunit
Beta; 60S Acidic Ribosomal Protein PO; Ezrin; Nucleophosmin;
Calreticulin; 14-3-3 Protein Gamma; Septin-11; Annexin A2;
ADP-Ribosylation Factor-Like Protein 2R; Heat Shock Cognate 71 kDa
Protein; Myosin Light Polypeptide 6; and Major Vault Protein.
[0045] According to particular non-limiting embodiments of the
invention, at least one, or at least two, or at least three, or at
least four, or at least five, or at least ten, optionally up to
five, up to ten, or up to twenty, or up to thirty, or up to forty,
or up to forty-six of said above-described secretory protein
biomarkers (where more than one biomarker is referred to as a
"panel") can be used to identify a high efficiency cell or cell
culture.
[0046] In certain embodiments, such panels include, but are not
limited to, one, two, three, four, five, six, seven, or all eight
of: EH-Domain Containing Protein 4; Heat Shock Protein Beta 1;
Myosin Regulatory Light Chain; Guanine Nucleotide Binding Protein
Beta 1; Translation Initiation Factor 3 Subunit 3; Vimentin; ATP
Synthase Subunit Beta; and Elongation Factor 1 Gamma. In certain of
such embodiments, EH-Domain Containing Protein 4; Heat Shock
Protein Beta 1; Myosin Regulatory Light Chain; and/or Guanine
Nucleotide Binding Protein Beta 1 are up-regulated, while
Translation Initiation Factor 3 Subunit 3; Vimentin; ATP Synthase
Subunit Beta; and/or Elongation Factor 1 Gamma are
down-regulated.
[0047] In certain embodiments, such panels include, but are not
limited to, one, two, three, four, five, six, seven, or all eight
of: EH-Domain Containing Protein 4; Heat Shock Protein Beta 1;
Myosin Regulatory Light Chain; Guanine Nucleotide Binding Protein
Beta 1; Translation Initiation Factor 3 Subunit 3; Vimentin; ATP
Synthase Subunit Beta; and/or Elongation Factor 1 Gamma. In certain
embodiments EH-Domain Containing Protein 4; Heat Shock Protein Beta
1; Myosin Regulatory Light Chain; and/or Guanine Nucleotide Binding
Protein Beta 1 are up-regulated, while Translation Initiation
Factor 3 Subunit 3; Vimentin; ATP Synthase Subunit Beta; and/or
Elongation Factor 1 Gamma are down-regulated.
[0048] In certain embodiments, such panels include, but are not
limited to, one, two, three, four, five, six, seven, or all eight
of: EH Domain-Containing Protein 4; 60S Acidic Ribosomal Protein
PO; Heterogeneous Nuclear Ribonucleoprotein K; Ezrin; ATP Synthase
Subunit Beta; Nucleophosmin; Vimentin; and/or Calreticulin. In
certain embodiments, EH Domain-Containing Protein 4; 60S Acidic
Ribosomal Protein PO; Heterogeneous Nuclear Ribonucleoprotein K;
and/or Ezrin are up-regulated, while ATP Synthase Subunit Beta;
Nucleophosmin; Vimentin; and/or Calreticulin are
down-regulated.
[0049] In certain embodiments, such panels include, but are not
limited to, one, two, three, four, five, six, or all seven of:
14-3-3 Protein Gamma; Protein Disulfide Isomerase A3 Precursor;
Septin-11; Annexin A2; ADP-Ribosylation Factor-Like Protein 2R;
Heat Shock Cognate 71 kDa Protein; and/or Myosin Light Polypeptide
6. In certain embodiments 14-3-3 Protein Gamma; Protein Disulfide
Isomerase A3 Precursor; Septin-11; Annexin A2; and/or
ADP-Ribosylation Factor-Like Protein 2R are up-regulated, while
Heat Shock Cognate 71 kDa Protein; and/or Myosin Light Polypeptide
6 are down-regulated.
[0050] In certain embodiments, such panels include, but are not
limited to, one, two, or all three of: Heat shock cognate 71 kDa
protein; Major Vault Protein; and/or Eukaryotic Translation
Initiation Factor5A-1. In certain embodiments, Heat shock cognate
71 kDa protein; Major Vault Protein; and/or Eukaryotic Translation
Initiation Factor5A-1 are down-regulated.
[0051] In certain embodiments, such panels include, but are not
limited to, one, two, or all three of: Guanine Nucleotide-Binding
Protein; Heat Shock Protein Beta-1; and/or 14-3-3 Protein
Zeta/Delta. In certain embodiments Guanine Nucleotide-Binding
Protein is down-regulated, while Heat Shock Protein Beta-1; and/or
14-3-3 Protein Zeta/Delta are up-regulated.
[0052] In certain embodiments, the expression level of the
biomarker, or biomarkers, being analyzed will be measured at a
specific point during the culture process. For example, but not by
way of limitation, the expression level of the biomarker, or
biomarkers, can be assayed on Day 1, Day 2, Day 3, Day 4, Day 5,
Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, Day 14,
or Day 15 of the cell culture process. In certain embodiments the
expression of the biomarker, or biomarkers, will be assayed at one,
two, three or more points during the cell culture process. In
certain embodiments, particular biomarkers need not all be assayed
at all time points, but rather certain biomarkers can be assayed at
certain time points and other biomarkers assayed at other, not
necessarily completely overlapping, time points.
[0053] In related, specific, non-limiting embodiments, the present
invention provides for kits for identifying modulation in
expression of one or a panel of said above-listed secretory protein
biomarkers. Such kids can, for example, comprise a detectable
antibody directed against said biomarker or biomarkers and a means
for detecting said antibody. In non-limiting specific embodiments,
in a panel provided in said kit, biomarkers listed above represent
up to about 20 percent, or up to about 30 percent, or up to about
50 percent, or up to about 75 percent, or up to about 90 percent,
or up to about 100 percent, of biomarkers in the entire panel to be
tested. Said kit may optionally further comprise one or more
positive and/or one or more negative control sample(s).
6.3 Methods of Using Biomarker Compositions
[0054] In certain embodiments of the present invention, one or more
protein biomarker, preferably one or more secretory protein
biomarker, is employed to identify a high efficiency cell or cell
culture. Such identification is often useful in the context of
commercial scale-up of recombinant protein production. For example,
but not by way of limitation, an initial, small scale cell culture
is initiated to promote the efficiency of recombinant protein
production. This can be facilitated by monitoring the expression
level of one or more secretory protein biomarker that is/are
associated with high efficiency expression of the protein of
interest. In certain embodiments, it is the up-regulation of a
secretory protein biomarker that is employed to identify a high
efficiency cell culture. In alternative embodiments it is the
down-regulation of a secretory protein biomarker that is employed
to identify a high efficiency cell culture. In preferred
embodiments, multiple secretory protein biomarkers are employed to
identify a high efficiency cell culture. In such embodiments where
multiple secretory protein biomarkers are employed, all of the
biomarkers can be up-regulated, all of the secretory protein
biomarkers can be down-regulated, or one or more of the secretory
protein biomarkers is up-regulated while one or more of the
secretory protein biomarkers is down-regulated
[0055] In alternative embodiments of the present invention, the
secretory protein biomarker is employed to monitor the expression
efficiency of an established large-scale cell culture, such as a
commercial cell culture. In such embodiments periodic monitoring of
one or more secreted protein biomarkers can ensure that the cell
culture is continuing to exhibit the high efficiency cell culture
phenotype. In certain examples, a change in the expression of the
biomarker can indicate a need to adjust one or more cell culture
conditions, such as, but not limited to, media type, temperature,
and pH. In certain embodiments it is the up-regulation of a
secretory protein biomarker that is employed to identify a high
efficiency cell culture. In alternative embodiments it is the
down-regulation of a secretory protein biomarker that is employed
to identify a high efficiency cell culture. In preferred
embodiments, multiple secretory protein biomarkers are employed to
identify a high efficiency cell culture. In such embodiments where
multiple secretory protein biomarkers are employed, all of the
biomarkers can be up-regulated, all of the secretory protein
biomarkers can be down-regulated, or one or more of the secretory
protein biomarkers.
6.4 High Efficiency Cells and Cell Cultures
[0056] The present invention further provides for a high efficiency
cell or cell culture which manifests a biomarker profile associated
with high efficiency protein expression. In particular, the present
invention provides for a high efficiency cell or cell culture which
manifests a secretory biomarker profile associated with high
efficiency protein expression. For example, and not by way of
limitation, said cell or cell culture exhibits a modulation of the
expression of one or more secretory protein biomarker set forth in
Tables 1-7.
[0057] According to particular non-limiting embodiments of the
invention, expression of at least one, or at least two, or at least
three, or at least four, or at least five, or at least ten of the
above-listed secretory biomarkers may be modulated in a cell or
cell culture of the invention. In a specific, non-limiting
embodiment of the invention, said cell is a CHO cell.
7. EXAMPLES
7.1 Example 1
7.1.1 Organelle Fractionation
[0058] Two Chinese Hamster Ovary (CHO) cell lines producing
recombinant glycoproteins of interest were employed throughout the
following examples. Both cell lines were shown to produce g/L
levels of the desired product, but that culture conditions
facilitating large increases in product titers (FIG. 1). Comparing
differences in secretory pathway protein levels using the same
clone but under these different culture conditions allowed for
findings as to what differences exist at the cellular level using
cells that have the same genetic makeup and gene amplification
levels.
[0059] To determine the relative distribution of candidate
biomarker secretory proteins inside the organelles of the
recombinant protein producer cell lines, it was necessary to first
purify these organelles. There are numerous well-established
protocols for organelle purification reported in the literature.
Typically, such protocols involve some form of cell disruption via
chemical or mechanical means followed by differential, or density
gradient centrifugation for purification and subsequent assessment.
Other methods include the use of cell disruption followed by
immunomagnetic separation via antibodies towards specific organelle
proteins (Vitale, N., K. Horiba, et al. (1998). "Localization of
ADP-ribosylation factor domain protein 1 (ARD1) in lysosomes and
Golgi apparatus." Proc Natl Acad Sci USA 95(15): 8613-8). In the
instant example, commercially available assay kits, which utilized
proprietary buffers and/or sucrose gradient ultracentrifugation to
purify both the ER and Golgi. The source cells for these
experiments came from 3 L benchtop Applikon bioreactors, from which
a large number of source cells were generated for organelle
fractionation. Any yield limitations of the assay were more than
compensated for by the large number of cells that were generated.
Verification of organelle enrichment was achieved using visual
verification by transmission electron microscopy (TEM) morphometry.
(FIG. 2).
7.1.2. Differential Mapping of the Secretory Process by
Two-Dimensional Gel Electrophoresis
[0060] The organelle enrichment described in Example 1 was followed
by 2D gel electrophoresis (2DGE) (FIG. 3). 2DGE is a
well-established analytical method that can resolve 1000-2000
proteins in complex mixtures by their isoelectric points in the
first dimension and then by their molecular weight using SDS-PAGE
in the second dimension. Detection of proteins that are run on 2D
gels can be accomplished by a number of different techniques,
including the silver staining technique employed in the gels
included in FIG. 3
[0061] Key objectives in 2-D analysis are to remove subjectivity,
to control variable gel running and gel image quality, to provide
high sensitivity for low level protein expression, and to identify
real changes in protein levels. Technology designed to meet these
challenges has been developed by Nonlinear Inc, including the
Progenesis software. For the instant samples, differential
expression was assigned using Progenesis software, followed by the
physical excision of spots for mass spectrometry identification.
The cut-off criteria used for the assignment of differential
expression was at least a 2-fold increase or decrease in normalized
spot volume intensity from the 2D gels on at least 1 of the time
course samples in either the ER or Golgi enriched fractions.
7.1.3. Mass Spectrometry (MS) Analysis of Differentially Expressed
Proteins
[0062] The protein spots identified and excised in Example 6.1.2.,
were then processed using Nano-flow ESI Q-TOF MS/MS analysis to
identify the particular biomarker secretory proteins. Indvidual
protein assigments were made using Spectrum Mill software
(Agilent), and the SwissProt database (FIG. 4). Table 1, in Section
6.2 above, lists the biomarker secretory proteins having
differential expression of at least a 2-fold increase or decrease
in normalized spot volume intensity from the 2D gels on at least 1
of the time course samples in either the ER or Golgi enriched
fractions that could be subsequently identified using mass
spectrometric analysis.
7.2. Example 2
7.2.1. Comparison of Three Distinct Protein-Producing Cell
Lines
[0063] This example describes a comparison of three distinct
protein producing cells lines cultured under media having differing
richness levels as well as a comparison of complex media versus
defined media. The cell culture, organelle isolation, protein
analysis, and protein identification was performed as described in
Example 1, sections 7.1.1.-7.1.3. above, unless described
otherwise.
7.2.2. Cell Line 1 Analysis
[0064] Cell Line 1 was cultured using two distinct process modes
and two distinct culture media. Culture 1 of Cell Line 1 employed a
fedbatch process mode and a complex, rich media. In contrast,
Culture 2 of Cell Line 1 employed a batch process mode and a
complex, lean media. The relative values (Culture 1/Culture 2) of
the product titer, specific productivity (q.sub.p), and integral of
viable cell density (IVC) for the Cell Line 1 comparison are
provided in FIG. 5. To identify differentially expressed proteins
samples of each culture were taken on Days 5, 8, and 11, and these
samples were analyzed by 2-D gel electrophoresis. Summary data
relating to the differentially expressed proteins is presented in
FIGS. 6 (Endoplasmic Reticulum Enriched Fraction) and 7 (Golgi
Enriched Fraction). Specific proteins identified as differentially
expressed are presented in Table 2 (Endoplasmic Reticulum Enrich
Fraction) and Table 3 (Golgi Enriched Fraction).
TABLE-US-00002 TABLE 2 Cell Line 1; Exemplary Differentially
Expressed Proteins - ER Fraction Relative Expression Representative
Proteins (Sample Day) Function EH-Domain Containing 0.94 (8)
Protein Transport Protein 4 Heat Shock Protein Beta 1 0.79 (8)
Cellular Stress Response Myosin Regulatory Light 0.61 (5) Cellular
Organization Chain Guanine Nucleotide Binding 0.56 (5) Signaling
Protein Beta 1 Translation Initiation -0.46 (8) Protein Translation
Factor 3 Subunit 3 Vimentin -0.70 (11) Cellular Organization ATP
Synthase Subunit Beta -0.70 (11) Metabolism Elongation Factor 1
Gamma -0.83 (8) Protein Translation
TABLE-US-00003 TABLE 3 Cell Line 1; Exemplary Differentially
Expressed Proteins - Golgi Fraction Relative Expression
Representative Proteins (Sample Day) Function EH-Domain Containing
0.94 (8) Protein Transport Protein 4 Heat Shock Protein Beta 1 0.79
(8) Cellular Stress Response Myosin Regulatory Light 0.61 (5)
Cellular Organization Chain Guanine Nucleotide Binding 0.56 (5)
Signaling Protein Beta 1 Translation Initiation -0.46 (8) Protein
Translation Factor 3 Subunit 3 Vimentin -0.70 (11) Cellular
Organization ATP Synthase Subunit Beta -0.70 (11) Metabolism
Elongation Factor 1 Gamma -0.83 (8) Protein Translation
7.2.3. Cell Line 2 Analysis
[0065] Cell Line 2 was cultured twice using the same batch process,
but with distinct culture media. Culture 1 of Cell Line 2 employed
a complex, rich media, while Culture 2 employed a complex, lean
media. The relative values (Culture 1/Culture 2) of the product
titer, specific productivity (q.sub.p), and integral of viable cell
density (IVC) for the Cell Line 2 comparison are provided in FIG.
8. To identify differentially expressed proteins samples of each
culture were taken on Days 8, and 11 (Endoplasmic Reticulum
Enriched Fractions) and Days 5, 8, and 11 (Golgi Enriched
Fractions), and these samples were analyzed by 2-D gel
electrophoresis. Summary data relating to the differentially
expressed proteins is presented in FIGS. 9 (Endoplasmic Reticulum
Enriched Fraction) and 10 (Golgi Enriched Fraction). Specific
proteins identified as differentially expressed are presented in
Table 4 (Endoplasmic Reticulum Enrich Fraction) and Table 5 (Golgi
Enriched Fraction).
TABLE-US-00004 TABLE 4 Cell Line 2; Exemplary Differentially
Expressed Proteins - ER Fraction Relative Expression Representative
Proteins (Sample Day) Function EH Domain-Containing 0.80 (8)
Protein Transport Protein 4 60S Acidic Ribosomal 0.72 (11) Protein
Translation Protein PO Heterogeneous Nuclear 0.60 (8) mRNA
Processing Ribonucleoprotein K Ezrin 0.58 (8) Cellular Organization
ATP Synthase Subunit Beta -0.41 (11) Metabolism Nucleophosmin -0.57
(11) Protein Translation Vimentin -0.73 (11) Cellular Organization
Calreticulin -1.13 (11) Protein Folding
TABLE-US-00005 TABLE 5 Cell Line 2; Exemplary Differentially
Expressed Proteins - Golgi Fraction Relative Expression
Representative Proteins (Sample Day) Function 14-3-3 Protein Gamma
0.69 (8) Signaling Protein Disulfide Isomerase 0.68 (5) Protein
Folding A3 Precursor Septin-11 0.66 (11) Cellular Organization
Annexin A2 0.58 (8) Signaling ADP-Ribosylation Factor-Like 0.46 (5)
Protein Processing Protein 2R Heat Shock Cognate 71 kDa -0.64 (5)
Protein Folding Protein Myosin Light Polypeptide 6 -0.67 (5)
Cellular Organization
7.2.4. Cell Line 3 Analysis
[0066] Cell Line 3 was cultured using two distinct process modes
and two distinct culture media. Culture 1 of Cell Line 3 employed a
batch process mode and a complex media. In contrast, Culture 2 of
Cell Line 3 employed a fedbatch process mode and a chemically
defined media. The relative values (Culture 1/Culture 2) of the
product titer, specific productivity (q.sub.p), and integral of
viable cell density (IVC) for the Cell Line 3 comparison are
provided in FIG. 11. To identify differentially expressed proteins
samples of each culture were taken on Days 5/6, 8/9, and 11/13, and
these samples were analyzed by 2-D gel electrophoresis. Summary
data relating to the differentially expressed proteins is presented
in FIGS. 12 (Endoplasmic Reticulum Enriched Fraction) and 13 (Golgi
Enriched Fraction). Specific proteins identified as differentially
expressed are presented in Table 6 (Endoplasmic Reticulum Enrich
Fraction) and Table 7 (Golgi Enriched Fraction).
TABLE-US-00006 TABLE 6 Cell Line 3; Exemplary Differentially
Expressed Proteins - ER Fraction Relative Expression Representative
Proteins (Sample Day) Function Heat shock cognate 71 kDa -1.41 (13)
Protein Folding protein Major Vault Protein -1.18 (9) Signaling
Eukaryotic Translation -0.94 (6) Protein Translation Initiation
Factor5A-1
TABLE-US-00007 TABLE 7 Cell Line 3; Exemplary Differentially
Expressed Proteins - Golgi Fraction Relative Expression
Representative Proteins (Sample Day) Function Guanine
Nucleotide-Binding -1.32 (9) Signaling Protein Heat Shock Protein
Beta-1 1.24 (9) Cellular Stress Response 14-3-3 Protein Zeta/Delta
0.96 (9) Signaling
[0067] All patents, patent applications, publications, product
descriptions and protocols, cited in this specification are hereby
incorporated by reference in their entirety. In case of a conflict
in terminology, the present disclosure controls.
[0068] While it will be apparent that the invention herein
described is well calculated to achieve the benefits and advantages
set forth above, the present invention is not to be limited in
scope by the specific embodiments described herein. It will be
appreciated that the invention is susceptible to modification,
variation and change without departing from the spirit thereof.
* * * * *