METHODS FOR HIGH-THROUGHPUT SCREENING OF CELL LINES
[0001] This Application claims the benefit of priority to U.S. Provisional Application
No. 60/793,991, filed April 21,2006, the specification of which is incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of pharmaceuticals. More
specifically, the invention pertains to methods for screening cell lines for the ability to produce
sufficient quantities and comparable quality of protein for industrial-scale production.
BACKGROUND OF THE INVENTION
[0003] High-throughput technology has become an important tool in pharmaceutical and
biotechnology research. High-throughput analytical methodologies utilize automated
procedures to rapidly analyze the activity of proteins, the level of protein expression, gene
expression, and the myriad of chemical interactions that occurs in a biological system. The
data generated by these methodologies and technologies has been put to use in a wide range of
fields such as cancer research, drug discovery, and crystallography (see, e.g., Abramovitz et al.
(2006) Proteome Sci. 4(1): 5 [Epub ahead of print]).
[0004] High-throughput analyses depend on the ability to sense a particular chemical
interaction or compound out of a vast array of chemical reactions occurring in a system. High-
throughput technologies have been used to probe the specific chemical interactions and levels
of expression of thousands of genes in a short period of time. To accomplish this difficult task,
certain techniques have been developed that utilize molecular signals (e.g., fluorophores) and
automated analyses that process information at a very rapid rate (see, e.g., Pinhasov et al.

(2004) Comb. Chem. High Throughput Screen. 7(2): 133-40). For example, microarray
technology has been extensively utilized to probe the interactions of thousands of genes at
once, while providing valuable information for specific genes (see, e.g., Mocellin and Rossi
(2007) Adv. Exp. Med. Biol. 593:19-30).
[0005] In the past few years, both automated and manual high-throughput protein
expression and purification has become an accessible means to rapidly screen and produce
soluble proteins for structural and functional studies (see Cabrita et al. (2006) BMC Biotechnol.
6:12). Developments such as filter-plate based assays for cloning, expression and purification,
ligation-independent cloning (LIC), and auto-induction for protein expression have been joined
with automated systems to create parallel production techniques of proteins that are simple and
cost-effective (see Cabrita et al. (2006) BMC Biotechnol. 6:12; Aslanidis et al. (1990) Nucleic
Acids Res. 18(20): 6069-6074). These approaches have provided researchers with powerful
tools to produce proteins at industrial-scale levels for use in pharmaceuticals and drug
development.
[0006] However, the purification and expression of proteins from particular cells presents
particular difficulties. Once a protein-of-interest has been identified, it is rather difficult to
obtain significant quantities of that protein in a purified form. As a result, cell lines producing
recombinant proteins are used to produce proteins in sufficient quantities for industrial
development.
[0007] Although cell lines can provide researchers with the opportunity to produce large
quantities of a particular protein, they are not always efficient producers of proteins. Certain
cell line clones may produce sub-optimal levels of protein. Other cell line clones may produce
optimal levels of protein expression, but fail to produce a fully functional pool of protein due to

either structural malformation or inappropriate post-translational modification. Such issues can
lead to wasted time and resources during development of a biologically relevant compound.
Therefore, high-throughput screening methodologies that rapidly and reliably provide
information on the quality and quantity of a protein expressed in a particular cell line are
needed. The present invention is directed to these and other important ends.
SUMMARY OF THE INVENTION
[0008] By analyzing the levels of expression of a protein-of-interest in a cell line prior to
starting full scale production of the protein-of-interest, significant time and resources will not
be wasted on cell lines that are of insufficient quality for high-throughput protein expression.
The present invention is based, in part, upon the discovery that high-throughput screening
procedures and high-throughput purification procedures can be performed to determine the cell
lines that produce sufficient quantities of a protein-of-interest. In addition, these procedures
can be utilized to screen the candidate cell lines to determine whether they produce the protein-
of-interest with a desired quality, for example, biological acivity. These high-throughput
screening and high-throughput-purification techniques (hereinafter together referred to as
"high-throughput screening") are therefore valuable methods for determining the proper cell
lines to utilize in industrial-scale protein expression. This discovery has been exploited to
provide an invention that allows for the use of high-throughput-screening methods to identify
cell lines that are useful for industrial-scale production of a protein-of-interest.
[0009] In one aspect, the invention provides a method of high-throughput screening of cell
lines for protein expression. The method includes the high-throughput titer screening of cell
lines to determine the level of protein expression in each cell line. Cell lines that produce a
desired level of protein expression are selected for subsequent high-throughput purification of

the protein. The cell line is selected for protein expression if the cell line produces a desired
level of protein expression and it has an appropriate quality.
[0010] In some embodiments, the protein is an antibody, ligand, receptor, subunit of
protein, fragment of protein, fusion protein, recombinant protein, and fragment of the same. In
certain embodiments, the protein is an antibody, a recombinant antibody, or a F(ab')2
fragment.
[0011] In other embodiments, the first binding agent is selected from the group consisting
of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant proteins,
and fragments of the same. In particular embodiments, the first binding agent is Protein A or
streptavidin. In still other embodiments, the binding agents can be attached to a solid support
such as beads, plates, and microarray chips. In many embodiments, the solid support
comprises cellulose, sepharose, polyacrylamide, glass, or polystyrene.
[0012] In other embodiments, the second binding agent is selected from the group
consisting of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant
proteins, and fragments of the same. In particular embodiments, the second binding agent is an
antibody and fragments of the same. In more particular embodiments, the antibody is a F(ab')2
fragment, and is even more particularly an F(ab')2 fragment that specifically binds to the Fc
portion of an antibody.
[0013] In still other embodiments, the detectable label is a fluorophore, chemical dye,
radioactive binding agent, chemiluminescent binding agent, electrochemiluminescent agent,
magnetic binding agent, paramagnetic binding agent, promagnetic binding agent, enzyme that
yield a colored product, enzyme that yield a chemiluminescent product, and enzyme that yield

a magnetic product. In very particular embodiments, the detectable label is ruthenium or
multiple ruthenium labels.
[0014] In some embodiments, the reagent comprises a resin, which, in particular
embodiments, has a third binding agent attached to it. In certain embodiments, the third
binding agent is selected from the group consisting of antibodies, ligands, receptors, fusion
proteins, subunits of proteins, recombinant proteins, and fragments of the same. While in
particular embodiments, the third binding agent is Protein A or streptavidin. In some
embodiments, the protein is eluted from the reagent using a vacuum. In other embodiments,
the protein is eluted by centrifugation. In still other embodiments, the protein is eluted by
gravity flow through the resin. In many embodiments, the screening of the incubated cell lines
utilizes an automated workstation.
[0015] In another aspect, the invention provides a method of high-throughput screening of
cell lines for protein expression. The method includes a step of incubating cell lines in media,
and contacting a solid support with a sample from each incubated cell line, fa this aspect, the
solid support has attached to its surface a first binding agent that binds to a protein in each
sample. The protein that is bound by the first binding agent is contacted with a second binding
agent, which is operably linked to a detectable label, that binds to the protein. The level of
protein expression in each sample is determined by detecting the label operably linked to the
second binding agent bound to the protein. The method also includes the selection of the cell
lines that have a desired level of protein expression. In some instances, this will be determined
by comparing the level of protein expression in each cell line to the average level of protein
expression in all of the cell lines. For instance, an increased level of protein expression as
compared to the average level of expression in all cell lines could be the desired level of

protein expression. Alternatively, a decreased level of protein expression could be the desired
level of protein expression, which would also be determined by comparing the level of protein
expression in each screened cell line to the average level of protein expression in all cell lines.
[0016] The method of this aspect of the invention further entails isolating supernatants
from the selected cell lines, and distributing each supernatant to a well of a multiwell plate.
The supernatants are then contacted with a reagent that binds to the protein. The protein is
eluted from the reagent and assayed for appropriate quality. According to this aspect of the
invention, a cell line is selected for protein expression if the cell line was selected in step e) and
the protein expressed by the cell line has appropriate quality. In some embodiments, the
protein is an antibody, ligand, receptor, subunit of protein, fragment of protein, fusion protein,
recombinant protein, and fragment of the same. In certain embodiments, the protein is an
antibody, a recombinant antibody, or a F(ab')2 fragment.
[0017] In other embodiments, the first binding agent is selected from the group consisting
of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant proteins,
and fragments of the same. In particular embodiments, the first binding agent is Protein A or
streptavidin. In still other embodiments, the binding agents can be attached to a solid support
such as beads, plates, and microarray chips. In many embodiments, the solid support
comprises cellulose, sepharose, polyacrylamide, glass, or polystyrene.
[0018] In other embodiments, the second binding agent is selected from the group
consisting of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant
proteins, and fragments of the same. In particular embodiments, the second binding agent is an
antibody and fragments of the same. In more particular embodiments, the antibody is a F(ab')2

fragment, and is even more particularly an F(ab')2 fragment that specifically binds to the Fc
portion of an antibody.
[0019] In still other embodiments, the detectable label is a fluorophore, chemical dye,
radioactive binding agent, chemiluminescent binding agent, electrochemiluminescent agent,
magnetic binding agent, paramagnetic binding agent, promagnetic binding agent, enzyme that
yield a colored product, enzyme that yield a chemiluminescent product, and enzyme that yield
a magnetic product. In very particular embodiments, the detectable label is ruthenium or
multiple ruthenium labels.
[0020] In some embodiments, the reagent comprises a resin, which, in particular
embodiments, has a third binding agent attached to it. In certain embodiments, the third
binding agent is selected from the group consisting of antibodies, ligands, receptors, fusion
proteins, subunits of proteins, recombinant proteins, and fragments of the same. While in
particular embodiments, the third binding agent is Protein A or streptavidin. In some
embodiments, the protein is eluted from the reagent using a vacuum. In many embodiments,
the screening of the incubated cell lines utilizes an automated workstation.
[0021] In another aspect, the invention provides a method of cell culture process
development. The method includes a step of incubating each cell line in a different condition
to be tested. Cell line samples are then placed into contact with a solid support from each cell
line from each different condition. In this aspect, the solid support has attached to its surface a
first binding agent that binds to a protein in each sample. The protein that is bound by the first
binding agent is contacted with a second binding agent, which is operably linked to a detectable
label, that binds to the protein. The level of protein expression in each sample is determined by
detecting the label operably linked to the second binding agent bound to the protein. The

method also includes the selection of the cell lines that have a desired level of protein
expression. In some instances, this will he determined by comparing the level of protein
expression in each cell line to the average level of protein expression in all of the cell lines.
For instance, an increased level of protein expression as compared to the average level of
expression in all cell lines could be the desired level of protein expression. Alternatively, a
decreased level of protein expression could be the desired level of protein expression, which
would also be determined by comparing the level of protein expression in each screened cell
line to the average level of protein expression in all cell lines.
[0022] The method of this aspect of the invention further entails isolating supernatants
from the selected cell lines, and distributing each supernatant to a well of a multiwell plate.
The supernatants are then contacted with a reagent that binds to the protein. The protein is
eluted from the reagent and assayed for appropriate quality. According to this aspect of the
invention, a proper cell culture condition is identified if the protein quantity and quality are
improved as compared to other conditions tested.
[0023] In other embodiments, the first binding agent is selected from the group consisting
of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant proteins,
and fragments of the same. In particular embodiments, the first binding agent is Protein A or
streptavidin. In still other embodiments, the binding agents can be attached to a solid support
such as beads, plates, and microarray chips. In many embodiments, the solid support
comprises cellulose, sepharose, polyacrylamide, glass, or polystyrene.
[0024] In other embodiments, the second binding agent is selected from the group
consisting of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant
proteins, and fragments of the same. In particular embodiments, the second binding agent is an

antibody and fragments of the same. In more particular embodiments, the antibody is a F(ab')2
fragment, and is even more particularly an F(ab')2 fragment that specifically binds to the Fc
portion of an antibody.
[0025] In still other embodiments, the detectable label is a fluorophore, chemical dye,
radioactive binding agent, chemiluminescent binding agent, electrochemiluminescent agent,
magnetic binding agent, paramagnetic binding agent, promagnetic binding agent, enzyme that
yield a colored product, enzyme that yield a chemiluminescent product, and enzyme that yield
a magnetic product. In very particular embodiments, the detectable label is ruthenium or
multiple ruthenium labels.
[0026] In some embodiments, the reagent comprises a resin, which, in particular
embodiments, has a third binding agent attached to it. In certain embodiments, the third
binding agent is selected from the group consisting of antibodies, ligands, receptors, fusion
proteins, subunits of proteins, recombinant proteins, and fragments of the same. While in
particular embodiments, the third binding agent is Protein A or streptavidin. In some
embodiments, the protein is eluted from the reagent using a vacuum. In many embodiments,
the screening of the incubated cell lines utilizes an automated workstation.
[0027] In certain embodiments, the condition to be tested is cell growth media. In other
embodiments, the condition to be tested is temperature. In still other embodiments, the
condition to be tested is humidity. In still more embodiments, the condition to be tested is
pressure. In yet more embodiments, the condition to be tested is oxygen pressure.
[0028] In yet another aspect, the invention provides a method for high-throughput
screening of cell lines for protein production. The method includes a first step of incubating
the cell lines in media. A sample from each cell line is placed onto a solid support, i.e., the

solid support is brought into contact with each sample. It should be noted that the solid support
has a first binding agent attached to its surface that binds to a protein in each sample. The first
binding agent binds the protein in the cell sample. The protein is contacted with a second
binding agent, which is operably linked to a detectable label, that binds to the protein. The
level of protein expression is determined in each sample by detecting the label operably linked
to the second binding agent bound to the protein. The cell lines are selected for protein
production based on whether the cell line has a desired level of protein expression as compared
to the average level of protein expression in the cell lines screened.
[0029] In some embodiments, the protein is an antibody, ligand, receptor, subunit of
protein, fragment of protein, fusion protein, recombinant protein, and fragment of the same. In
certain embodiments, the protein is an antibody, a recombinant antibody, or a F(ab')2
fragment.
[0030] In other embodiments, the first binding agent is selected from the group consisting
of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant proteins,
and fragments of the same. In particular embodiments, me first binding agent is Protein A or
streptavidin. In still other embodiments, the binding agents can be attached to a solid support
such as beads, plates, and microarray chips. In many embodiments, the solid support
comprises cellulose, sepharose, polyacrylamide, glass, or polystyrene.
[0031] In other embodiments, the second binding agent is selected from the group
consisting of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant
proteins, and fragments of the same. In particular embodiments, the second binding agent is an
antibody and fragments of the same. In more particular embodiments, the antibody is a F(ab')2

fragment, and is even more particularly an F(ab')2 fragment that specifically binds to the Fc
portion of an antibody.
[0032] In still other embodiments, the detectable label is a fluorophore, chemical dye,
radioactive binding agent, chemiluminescent binding agent, electrochemiluminescent agent,
magnetic binding agent, paramagnetic binding agent, promagnetic binding agent, enzyme that
yield a colored product, enzyme that yield a chemiluminescent product, and enzyme that yield
a magnetic product In very particular embodiments, the detectable label is ruthenium or
multiple ruthenium labels. In certain embodiments, the method further comprises a sialic acid
assay.
BRIEF DESCRIPTION OF THE FIGURES
[0033] The foregoing and other objects of the present invention, the various features
thereof, as well as the invention itself may be more fully understood from the following
description, when read together with the accompanying drawings in which:
[0034] Figure 1 is a pictorial representation of a high-throughput protein expression assay
showing the detection of an antibody in a cell sample.
[0035] Figure 2 is a graphical representation of a high-throughput screening assay showing
the signal-to-background ratio of using various F(ab')2 fragments conjugated to ruthenium.
[0036] Figure 3 is a graphical representation of a high-throughput screening assay showing
the signal-to-background ratio of using various F(ab')2 fragments conjugated to ruthenium.
[0037] Figure 4 is a graphical representation of a plot showing the sensitivity of high-
throughput screening assays detecting GPlbα, IL13 receptor, and TNFR fusion protein at
different concentrations.

[0038] Figure 5 is a graphical representation of a plot showing the sensitivity of high-
throughput screening assays detecting anti-GDF8, anti-CD22, and anti-Lewis Y antibodies at
different concentrations.
[0039] Figure 6 is a graphical representation of a plot showing the assay time required for
the high-throughput titer screening assay and a HPLC assay.
[0040] Figure 7 is a graphical representation showing a comparison between the high-
throughput screening assay and HPLC at determining the levels of TNFR fusion protein in
samples.
[0041] Figure 8 is a graphical representation showing the levels of expression in clones as
determined by a high-throughput titer screening assay.
[0042] Figure 9 is a graphical representation showing a comparison between the high-
throughput screening assay and HPLC at determining the levels of anti-Lewis Y antibodies in
samples.
[0043] Figure 10 is a graphical representation showing a comparison between the high-
throughput screening assay and HPLC at determining the levels of PSGL and GPlbot in
samples.
[0044] Figure 11 is a graphical representation showing a comparison between the high-
throughput screening assay and HPLC at determining the levels of anti-Aβ in samples.
[0045] Figure 12 is a graphical representation showing the percentage of high molecular
weight protein found in samples purified using different purification procedures.
[0046] Figure 13 is a graphical representation showing the percentage of high molecular
weight protein found in samples purified using different purification procedures.

[0047] Figure 14 is a graphical representation of a bar graph showing the amount of high
molecular weight protein found in samples isolated from different cell lines grown in different
conditions.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The patent and scientific literature referred to herein establishes knowledge that is
available to those of skill in the art. The issued US patents, allowed applications, published
foreign applications, and references, including GenBank database sequences, that are cited
herein are hereby incorporated by reference to the same extent as if each was specifically and
individually indicated to be incorporated by reference.
1.1. General
[0049] An embodiment of the present invention in part provides methods of screening cell
lines for the capability to produce a protein-of-interest. The invention also describes processes
for improved efficiency in the industrial-scale production of proteins for pharmaceutical and
biological studies. In particular, the present invention allows for the efficient production of
proteins that can be utilized in pharmaceutical treatments of diseases such as cancer,
Alzheimer's Disease, and diabetes. Furthermore, embodiments of the invention provide a
method for high-throughput screening of cell lines to determine the quantity and quality of a
protein-of-interest produced by the cell lines.
[0050] Accordingly, one aspect of the invention provides a method of high-throughput
screening of cell lines for protein expression. The method utilizes a first binding agent that
binds to the protein-of-interest and a second binding agent that is operably linked to a

detectable label that binds to the protein-of-interest. In some embodiments, the first binding
agent is attached to a solid support such as a bead, magnetic bead, a plate, or a microarray chip.
The method also uses a reagent that binds to the protein-of-interest, and allows for the protein
to be purified from a sample derived from the cell line. In some embodiments of the invention,
the protein is purified from the reagent by means of a vacuum drawing the eluted protein from
the reagent and through a filter. In still other embodiments, the solution is allowed to flow
through the reagent by gravity flow.
[0051] As used here, the term 'therapeutic protein" is a protein or peptide that has a
biological effect on a region in the body on which it acts or on a region of the body on which it
remotely acts via intermediates. A therapeutic protein can be, for example, a secreted protein,
such as, an antibody, an antigen-binding fragment of an antibody, a soluble receptor, a receptor
fusion, a cytokine, a growth factor, an enzyme, or a clotting factor, as described in more detail
herein below. The above list of proteins is merely exemplary in nature, and is not intended to
be a limiting recitation. One of ordinary skill in the art will understand that any protein may be
used in accordance with the present invention and will be able to select the particular protein to
be produced based as needed.
[0052] As used in the specification, the terms polypeptide, protein and peptide are
synonymous and are used interchangeably. Accordingly, as used herein, the size of a protein,
peptide or polypeptide generally comprises more than 2 amino acids. For example, a protein,
peptide or polypeptide can comprise from about 2 to about 20 amino acids, from about 20 to
about 40 amino acids, from about 40 to about 100 amino acids, from about 100 amino acids to
about 200 amino acids, from about 200 amino acids to about 300 amino acids, and so on.

As used herein, an amino acid refers to any naturally occurring amino acid, any amino acid
derivative or any amino acid mimic known in the art. In certain embodiments, the residues of
the protein or peptide are sequential, without any non-amino acid interrupting the sequence of
amino acid residues. In other embodiments, the sequence may comprise one or more non-
amino acid moieties. In particular embodiments, the sequence of residues of the protein or
peptide may be interrupted by one or more non-amino acid moieties.
[0053] As used herein, term "antibody" is used to refer to any antibody-like molecule that
has an antigen binding region, and includes antibody fragments such as Fab1, Fab, F(ab').sub.2,
single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. Techniques for
preparing and using various antibody-based constructs and fragments are well known in the art.
Means for preparing and characterizing antibodies are also well known in the art (See, e.g.,
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988;
incorporated herein by reference). For example, an antibody can include at least one, and
preferably two full-length heavy chains, and at least one, and preferably two light chains. The
term "antibody" as used herein includes an antibody fragment or a variant molecule such as an
antigen-binding fragment (e.g., an Fab, F(ab')2, Fv, a single chain Fv fragment, a heavy chain
fragment (e.g., a camelid VHH) and a binding domain-immunoglobulin fusion (e.g., SMIP™).
The antibody can be a monoclonal or single-specificity antibody. The antibody can also be a
human, humanized, chimeric, CDR-grafted, or in vitro generated antibody. In yet other
embodiments, the antibody has a heavy chain constant region chosen from, e.g., IgGl, IgG2,
IgG3, or IgG4. In another embodiment, the antibody has a light chain chosen from, e.g., kappa
or lambda. In one embodiment, the constant region is altered, e.g., mutated, to modify the
properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding,

antibody glycosylation, the number of cysteine residues, effector cell function, or complement
function). Typically, the antibody specifically binds to a predetermined antigen, e.g., an
antigen associated with a disorder, e.g., a neurodegenerative, metabolic, inflammatory,
autoimmune and/or a malignant disorder.
[0054] Small Modular ImmunoPharmaceuticals (SMIP™) provide an example of a variant
molecule comprising a binding domain polypeptide. SMIPs and their uses and applications are
disclosed in, e.g., U.S. Published Patent Application. Nos. 2003/0118592,2003/0133939,
2004/0058445, 2005/0136049, 2005/0175614, 2005/0180970,2005/0186216,2005/0202012,
2005/0202023, 2005/0202028, 2005/0202534, and 2005/0238646, and related patent family
members thereof, all of which are hereby incorporated by reference herein in their entireties.
Single domain antibodies can include antibodies whose complementary determining regions
are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain
antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from
conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than
those derived from antibodies. Single domain antibodies may be any of the art, or any future
single domain antibodies. Single domain antibodies may be derived from any species
including, but not limited to mouse, human, camel, Ilama, goat, rabbit, bovine. According to
one aspect of the invention, a single domain antibody as used herein is a naturally occurring
single domain antibody known as heavy chain antibody devoid of light chains. Such single
domain antibodies are disclosed in WO 9404678 for example. For clarity reasons, this variable
domain derived from a heavy chain antibody naturally devoid of light chain is known herein as
a VHH or nanobody to distinguish it from the conventional VH of four chain
immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae

species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides
Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are
within the scope of the invention.
[0055] Examples of binding fragments encompassed within the term "antigen-binding
fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the
VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two
Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of
the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single
arm of an antibody, (v) a dAb fragment, which consists of a VH domain; (vi) a camelid or
camelized variable domain, e.g., a VHH domain; (vii) a single chain Fv (scFv); (viii) a
bispecific antibody; and (ix) one or more fragments of an immunoglobulin molecule fused to
an Fc region. Furthermore, although the two domains of the Fv fragment, VL and VH, are
coded for by separate genes, they can be joined, using recombinant methods, by a synthetic
linker that enables them to be made as a single protein chain in which the VL and VH regions
pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al.
(1988) Science 242:423-26; Huston et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879-83).
Such single chain antibodies are also intended to be encompassed within the term "antigen-
binding fragment" of an antibody. These antibody fragments are obtained using conventional
techniques known to those skilled in the art, and the fragments are evaluated for function in the
same manner as are intact antibodies.
[0056] Other than "bispecific" or "bifunctional" antibodies, an antibody is understood to
have each of its binding sites identical. A "bispecific" or "bifunctional antibody" is an artificial
hybrid antibody having two different heavy/light chain pairs and two different binding sites.

Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas
or linking of Fab1 fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-
321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).
[0057] As used herein, the term "cell line" means a cell that is maintained in culture and
has acquired the ability to grow in ex vivo conditions. Cell lines can be either immortalized or
transiently established as "primary cell lines." In certain embodiments, cell lines are
established by techniques known in the art (see, e.g., Kwak et al. (2006) Anim. Biotechnol.
17(1): 51-8). In some embodiments, the cell lines are antibody-producing cells, which can be
produced by techniques known in the art (see, e.g., Dessain et al. (2004) J. Immunol. Methods.
291(1-2): 109-22.). The cell lines can also be obtained from commercial sources such as
ATCC cell biology collections (American Type Culture Collections, Mannassas, VA).
[0058] As used herein, the term "binding agent" means a molecule that can associate with
any other molecule by way of covalent bonds, hydrogen bonds, ionic bonds, Van der Waals
forces, London forces, or any combination of the forces. Binding agents include, but are not
limited to, proteins and fragments thereof, peptidomimetic compounds, antibodies and
fragments thereof, nucleic acids, toxins, and small molecules.
[0059] Binding agents can be disposed on a derivatized solid support through methods well
known in the art. Solid supports include, but are not limited to, beads, magnetic beads,
microarray chips, nitrocellulose membranes, nylon membranes, multiwell plates, and PVDF
membranes. In some embodiments, the solid support is a plate in which an electrode is
disposed beneath the plate, which creates a magnetic field that attracts a binding agent bound to
a magnetic material. The plates are used in accordance with manufacturer's protocols (see
Meso Scale Discovery, Gaithersburg, MD).

[0060] Solid supports can be composed of glass, polystyrene, plastic, magnetic metals such
as iron, polyacrylamide, sepharose, cellulose, or any inert support that does not affect the
binding agents ability to bind a protein. Solid supports are obtained commercially from, e.g.,
Applied Biosystems, (Foster City, CA).
[0061] Moreover, in some embodiments, binding agents are disposed on a solid support
such as a microarray chip utilizing methods practiced by those of ordinary skill in the art
through a process called "printing" (see, e.g., Schena et. al., (1995) Science, 270(5235): 467-
470). The term "printing," as used herein, refers to the placement of spots onto the solid
support in such close proximity as to allow a maximum number of spots to be disposed onto a
solid support. The printing process can be carried out by, e.g., a robotic printer. The
VersArray CHIP Writer Prosystem (BioRad Laboratories) using Stealth Micro Spotting Pins
(Telechem International, Inc, Sunnyvale, CA) is a non-limiting example of a chip-printing
device that can be used to produce the focused microarray for this aspect
[0062] As used herein, the term "appropriate quality" means the quality that is particularly
related to the protein-of-interest An appropriate quality includes, but is not limited to,
enzymatic reactions, antibody-epitope interactions, and nucleic acid-protein interactions. An
appropriate quality can be assayed by analyzing a particular physicochemical aspect of the
protein-of-interest. For instance, appropriate quality can be, without intending to limit the
types of assays that can be used, related to the size, charge, carbohydrate content of the protein,
binding activity, and enzymatic activity. Physical structure analyses such as NMR can be used
to determine the overall tertiary and secondary structure of the protein. In addition, lectin-
based assays and sialic acid assays, which will be described more fully below, can be used to
determine the physicochemical aspects of the protein-of-interest. In other words, appropriate

quality can be determined by looking not only to the chemical activities of the protein-of-
interest, but to the physical structure of the protein-of-interest as well.
[0063] As used herein, the term "elute" means to extract from a resin or binding agent by
the use of a solvent. The process of eluting a protein-of-interest from a resin or binding agent
can involve a solution that contains a molecule capable of dislodging the protein-of-interest
from the binding agent. In addition, the solution can have a pH mat alters the binding
characteristics of the resin or binding agent such that the protein-of-interest no longer
associates with the protein-of-interest. It should be noted that any solution can be used to elute
a protein in the present invention so long as the process does not affect the overall functionality
or quality of the protein-of-interest.
[0064] As used herein, the term "resin" means any solid or semi-solid organic products of
natural or synthetic origin. Resins include any material that can be conjugated to a moiety that
allows for purification of a molecule from a complex mixture. Moieties useful in the present
invention include cationic molecules, anionic molecules, metals, metalloids, polysaccharides,
polypeptides, proteins, nucleic acids, peptides, small organic molecules, and peptidomimetic
compounds. In particular, resins can themselves be composed of any inert compound
including, but not limited to, sephadex, polystyrene, polyacrylamide, or neutral
polysaccharides. Resins can be commercially obtained from, e.g., Clontech Laboratories, Inc.
(Mountain View, CA).
[0065] The term "high-throughput" as used herein means allowing for a fast and simple
methodology to determine the presence of desirable protein expression in a cell line. Desirable
protein expression refers to both the quantity and quality of the biological molecules expressed
by the cell lines screened by high-throughput techniques. High-throughput methodology also

can include automated systems for processing the biological molecules and automated data
processing for large-scale screening.
[0066] As used herein, the term "high-throughput titer screening" means a procedure used
to determine the quantity of protein expressed by a cell. High-throughput titer screening
includes the use of binding agents to identify a protein-of-interest in a sample derived from a
cell. The binding agents can be operably linked to a detectable label or a solid support, all of
which will be described more fully below.
[0067] As used herein, the term "high-throughput purification" means the process of
purifying proteins from multiple cell samples at the same or substantially same time.
[0068] As used herein, the term "cell culture process development" means procedures to
bring about the optimization of the conditions necessary to produce proteins at sufficient
quantities and qualities for industrial or small-scale production. The conditions that can be
tested using cell culture process development include, but are not limited to, salt concentration,
media content, growth temperature, atmospheric pressure, atmospheric oxygen content, culture
agitation, and carbon dioxide content. The cell culture process development includes the steps
of determining the quantity of a protein (high-throughput titer screening) and the quality of a
protein (high-throughput purification).
[0069] Methods of determining the quality of a protein for the purposes of cell culture
process development include, but are not limited to, binding assays, lectin assays, sialic acid
assays, NMR, circular dichroism, mass spectrometry, MALDI-TOF, enzymatic assays,
colorimetric assays, and amino acid sequencing. These assays can be used at any time during
the cell culture process development method. In certain embodiments, the assays are
performed after the completion of the high-throughput purification of the protein-of-interest.

[0070] The term "protein-of-interest" as used herein means any protein, or protein-like
molecule, produced for biological, medical, medicinal, or pharmaceutical purposes. For
example, a protein-of-interest can be a therapeutic protein. Proteins-of-interest can be
produced from nucleic acid sequences, whether chromosomal or extrachromosomal, which
include, but are not limited to, pre-messenger RNA, messenger RNA, transfer RNA,
heteronuclear RNA ("HnRNA"), ribosomal RNA, single-stranded DNA, and double-stranded
RNA. Extrachromosomal sources of nucleic acid sequences can include double-strand DNA
viral genomes, single-stranded DNA viral genomes, double-stranded RNA viral genomes,
single-stranded RNA viral genomes, bacterial DNA, mitochondrial genomic DNA, cDNA or
any other foreign source of nucleic acid that is capable of generating a protein-of-interest. A
protein-of-interest can be any structure or combination of structures. For example, proteins-of-
interest include, but are not limited to, recombinant proteins, proteins containing a quaternary
structure, glycosylated proteins, lipidated proteins, oligopeptides, peptides, protein domains,
protein subunits, antibodies or fragments thereof, and antibody-like molecules. Proteins-bf-
interest also include, for example, fusion proteins. Fusion proteins generally have all or a
substantial portion of a targeting peptide, linked at the N- or C-terminus, to all or a portion of a
second polypeptide or protein. For example, fusions may employ leader sequences from other
species to permit the recombinant expression of a protein in a heterologous host. Another
useful fusion includes the addition of an immunologically active domain, such as an antibody
epitope, to facilitate purification of the fusion protein. A fusion protein can include a targeting
moiety, e.g., a soluble receptor fragment or a ligand, and an immunoglobulin chain, an Fc
fragment, a heavy chain constant regions of the various isotypes, including: IgGl, IgG2, IgG3,
IgG4, IgM, IgAl, IgA2, IgD, and IgE). For example, the fusion protein can include the

extracellular domain of a receptor, and, e.g., fused to, a human immunoglobulin Fc chain (e.g.,
human IgG, e.g., human IgGl or human IgG4, or a mutated form thereof), m one embodiment,
the human Fc sequence has been mutated at one or more amino acids, e.g., mutated at residues
254 and 257 from the wild type sequence to reduce Fc receptor binding. The fusion proteins
may additionally include a linker sequence joining the first moiety to the second moiety, e.g.,
the immunoglobulin fragment. For example, the fusion protein can include a peptide linker,
e.g., a peptide linker of about 4 to 20, more preferably, 5 to 10, amino acids in length; the
peptide linker is 8 amino acids in length. For example, the fusion protein can include a peptide
linker having the formula (Ser-Gly-Gly-Gly-Gly)y wherein y is 1,2, 3, 4, 5, 6, 7, or 8. In other
embodiments, additional amino acid sequences can be added to the N- or C-terminus of the
fusion protein to facilitate expression, steric flexibility, detection and/or isolation or
purification.
[0071] Inclusion of a cleavage site at or near the fusion junction will facilitate removal of
the extraneous polypeptide after purification. Other useful fusions include linking of functional
domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals
or transmembrane regions. Examples of proteins or peptides feat may be incorporated into a
fusion protein include cytostatic proteins, cytocidal proteins, pro-apoptosis agents, anti-
angiogenic agents, hormones, cytokines, growth factors, peptide drugs, antibodies, Fab
fragments antibodies, antigens, receptor proteins, enzymes, lectins, MHC proteins, cell
adhesion proteins and binding proteins. Methods of generating fusion proteins are well known
to those of skill in the art. Such proteins can be produced, for example, by chemical attachment
using bifunctional cross-linking reagents, by de novo synthesis of the complete fusion protein,

or by attachment of a DNA sequence encoding the targeting peptide to a DNA sequence
encoding the second peptide or protein, followed by expression of the intact fusion protein.
[0072] In certain embodiments, a fusion protein is a tumor necrosis factor inhibitor, for
example in the form of tumor necrosis factor alpha and beta receptors (TNFR-1; EP 417,563
published Mar. 20,1991; and TNFR-2, EP 417,014 published Mar. 20,1991, each of which is
incorporated herein by reference in its entirety), and is analysed in accordance with the present
invention (for review, see Naismith and Sprang, J Inflamm. 47(l-2):l-7, 1995-96, incorporated
herein by reference in its entirety). According to some embodiments, a tumor necrosis factor
inhibitor comprises a soluble TNF receptor. In certain embodiments, a tumor necrosis factor
inhibitor comprises a soluble TNFR fused to any portion of an immunoglobulin protein,
including the Fc region of an immunoglobulin. In certain embodiments, TNF inhibitors of the
present invention are soluble forms of TNFR I and TNFR II. In certain embodiments, TNF
inhibitors of the present invention are soluble TNF binding proteins. In certain embodiments,
the TNF inhibitors of the present invention are TNFR-Fc, for example, etanereept. As used
herein, "etanercept," refers to a TNFR-Fc, which is a dimer of two molecules of the
extracellular portion of the p75 TNF-α receptor, each molecule consisting of a 235 amino acid
Fc portion of human IgGl. In accordance with the invention, an anti-senescence compound,
such as carnosine, is used to decrease the amount of misfolded and/or aggregated protein
during the production of TNFR-Fc.
[0073] Proteins or peptides may be made by any technique known to those of skill in the
art, including the expression of proteins, polypeptides or peptides through standard molecular
biological techniques, the isolation of proteins or peptides from natural sources, or the chemical
synthesis of proteins or peptides. The coding regions for known genes may be amplified and/or

expressed using the techniques disclosed herein or as would be know to those of ordinary skill
in the art (see, e.g., Kaleeba et al. (2006) Science 311 (5769):1921-4). Alternatively, various
commercial preparations of proteins, polypeptides and peptides are known to those of skill in
the art.
[0074] Furthermore, as used herein, the term "desired level of expression" means the
quantity of protein necessary, depending on the physicochemical characteristics of the protein,
to allow for subsequent purification of the protein. The desired levels of expression of a
protein-of-interest are dependent on a number of factors related to the production methods to be
utilized. For instance, the level of expression from a particular cell line allows for protein
quality and quantity analysis as well as efficient purification of the protein. The desired level
of protein expression from a cell line, therefore, is determined by one of skill in the art in view
of the protein's characteristics and the purification and assay methods to be used on the protein.
[0075] In certain embodiments, the desired levels of protein expression are selected based
on the particular requirements for the protein-of-interest. For instance, one of skill in the art
can choose a desired level of protein expression to be an increased level of expression of a
protein-of-interest to maximize the amount of protein produced. In other embodiments, a
decreased level of expression of the protein-of-interest is chosen to be the desired level of
protein expression in the case of proteins, such as a toxin, that are toxic at high levels in the cell
producing the toxin. In addition, a decreased level of protein expression is selected in
situations when the protein-of-interest forms inclusion bodies at high concentration levels.
Therefore, the level of protein expression selected by one of skill in the art depends on the
characteristics of the protein-of-interest.

[0076] In determining the quantity and quality of a protein produced by a cell line, a cell
line sample is typically necessary for assessment of protein expression and protein quality. In
certain embodiments, a cell line sample is isolated using means that are known in the art such
as cell lysis and supernatant isolation (see, e.g., Vara et al. (2005) Biomaterials 26(18): 3987-
93; Iyer et al. (1998) J. Biol. Chem. 273(5):2692-7). Alternatively, cell line samples are
isolated from a medium that has a secreted protein such as an antibody, extracellular matrix
protein, or serum protein. In such embodiments, the medium is the sample that will be tested
for protein quantity and quality. In one embodiment, the medium sample is used in an assay to
test for protein quantity, in the absence of any prior purification steps, as further described
herein.
[0077] Another aspect of the invention provides a method of high-throughput screening of
cell lines for protein production. In this method, the levels of protein expression are
determined by contacting a solid support with a cell line sample containing a protein. In one
embodiment, the cell line sample is cell culture media. The solid support has attached to its
surface a first binding agent mat is capable of binding to the protein. The method also includes
a second binding agent that binds to the protein bound by the first binding agent and
immobilized on the solid support. The second binding agent is operably linked to a detectable
label. The cell lines are selected based on the desired level of expression required for the
protein.
[0078] The level of expression of a particular protein can be measured by "dot blot" in
which a first binding agent is immobilized on a membrane such as nitrocellulose, nylon, or
PVDF (see, e.g., Heinicke et al. (1992) J. Immunol. Methods. 152(2): 227-36.). Protein
microarray technology can also be used to determine the expression of proteins in a sample.

Alternatively, a sample is placed into a well of a multiwell plate that contains the first binding
agent. In such embodiments, similar techniques such as ELISA analysis are routine in the art
(see, e.g., Ausubel, et al. (1996)Current Protocols in Molecular Biology, Vol. 1, pp. 4.2.1-
4.2.9, John Wiley & Sons, Inc.
[0079] In some embodiments, the high-throughput screening and/or purification of the cell
line samples are performed using an automated workstation. In addition, the cell culture
process development can be performed using automated workstations. Automated
workstations are commonly utilized in the art to perform many experiments in a short period of
time. Examples of automated workstations include, but are not limited to, the TECAN Genesis
Workstation (TECAN Schweiz AG, Mannedorf, CH) and the Biomek FX Workstation
(Beckman Coulter, Fullerton, CA). Methods of use can be obtained from the manufacturers of
automated workstations and are well known in the art
1.2. Binding Agents
[0080] Aspects of the invention utilize binding agents to bind a protein-of-interest. In
certain embodiments, a binding agent is an antibody or fragment thereof. Where the binding
agent that specifically binds a protein is an antibody, the antibody may be, without limitation, a
polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a
genetically engineered antibody, a bispecific antibody (where one of the specificities of the
bispecific antibody specifically binds to the triosephosphate isomerase protein), antibody
fragments (including but not limited to "Fv," "F(ab')2," "F(ab)," and "Dab"); and single chains
representing the reactive portion of an antibody ("SC-MAb"). Methods for making antibodies
and other binding agents are well known (see, e.g., Coligan et al. (1991) Current Protocols in

Immunology, John Wiley and Sons, Inc.; Jones et al. (1986) Nature 321: 522-525; Marx (1985)
Science 229: 455-456; Rodwell (1989) Nature 342: 99-100; Clackson (1991) Br. J. Rheumatol
3052: 36-39; Reichman et al. (1988) Nature 332: 323-327; Verhoeyen, etal. (1988) Science
239: 1534-1536).
[0081] The binding agent can be an antibody or fragment thereof that binds to the Fc
portion of an antibody. In certain embodiments, the binding agent allows for detection of
antibodies in a cell line sample. In particular embodiments, an antibody, which is the second
binding agent, is detectably labeled with an electrochemiluminescent detectable label such as
ruthenium. In addition, the second binding agent can be a F(ab')2 fragment detectably labeled
with an electrochemiluminescent detectable label such as ruthenium.
[0082] The detection of a protein of interest using F(ab')2 fragments is shown in Figure 1.
The F(ab')2 fragment is operably linked to a detectable label such as an Ori-Tag. In Figure 1,
the F(ab')2 fragment recognizes the Fc portion of an antibody, which is a protein-of-interest in
this pictorial example. The antibody has been immobilized on a bead, which has Protein A or
streptavidin conjugated to it (Fig. 1). The binding of the F(ab')2 fragment is observed as a
generation of light (Fig. 1).
[0083] It is important to note that the antibodies used as a first binding agent in an aspect of
the present invention can be coupled to the surface of a solid support. Coupling of the first
binding agent improves the signal strength of the reaction and produces improved results.
Common coupling agents include, but are not limited to, silanization using (3-mercaptopropyl)
trimethoxysilane, agarose coating, and poly-L-lysine films. Additionally, recombinant
antibodies can be engineered to include a tag facilitating coupling to the support For example,
a recombinant antibody having a histidine tag can be coupled to supports coated with nickel.

[0084] In addition, compounds such as peptides, peptidomimetic compounds, and small
molecules can be used as binding agents. Binding agents can be synthesized from peptides or
other biomolecules, including, but not limited, to saccharides, fatty acids, sterols, isoprenoids,
purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof
and the like. Phage display libraries and chemical combinatorial libraries can be used to
develop and select synthetic compounds that are acceptable binding agents for a protein-of-
interest. Also envisioned in the invention is the use of potential binding agents made from
peptoids, random bio-oligomers (U.S. Pat. No. 5,650,489), benzodiazepines, diversomeres such
as dydantoins, benzodiazepines and dipeptides, nonpeptidal peptidomimetics with a beta-D-
glucose scaffolding, oligocarbamates or peptidyl phosphonates.
[0085] In certain examples, binding agents can be peptides that are designed to specifically
interact, bind, or associate with a protein. Peptide binding agents can also interact, associate,
or bind with an amino acid sequence of any other protein. Peptides can be subjected to directed
or random chemical modifications such as acylation, alkylation, esterification, amidification,
etc.
[0086] Identification and screening of peptide binding agents is further facilitated by
determining structural features of theprotein-of-interest, e.g., using X-ray crystallography,
neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for
structure determination. Computer algorithms can further facilitate binding agent
identification. Such computer algorithms are employed mat are capable of scanning a database
of peptides and small molecules of known three-dimensional structure for candidates that fit
geometrically into the target protein's site (see, e.g., Chen and Kellogg (2005) J. Comput.
Aided Mol. Des. 19(2):69-82). Most algorithms of this type provide a method for finding a

wide assortment of chemical structures that are complementary to the shape of a binding pocket
or region of a domain of a protein. Each of a set of peptides from a particular database can be
compared to determine the particular peptides that have the most potential for interacting with a
protein-of-interest.
[0087] The compounds of the present invention can also be peptidomimetic compounds
that can be at least partially unnatural. The peptidomimetic compound can be a small molecule
mimic of a portion of any desirable amino acid sequence. The compound can have increased
stability, efficacy, potency and bioavailability by virtue of the mimic. Further, the compound
can have decreased toxicity. The peptidomimetic compound can have enhanced mucosal
intestinal permeability. The compound can be synthetically prepared. The compound of the
present invention can include L-,D- or unnatural amino acids, alpha, alpha-disubstituted amino
acids, N-alkyl amino acids, lactic acid (an isoelectronic analog of alanine). The peptide
backbone of the compound can have at least one bond replaced with PSI-[CH=CH] (Kempf et
al. (1991) Int. J. Pept. Protein Res. 38(3): 237-41). The compound can further include
trifluorotyrosine, p-Cl-phenylalanine, p-Br-phenylalanine, poly-L-propargylglycine, poly-D,L-
allyl glycine, or poly-L-allyl glycine.
[0088] One example of the present invention is a peptidomimetic compound wherein the
compound has a bond, a peptide backbone or an amino acid component replaced with a suitable
mimic. Examples of unnatural amino acids which can be suitable amino acid mimics include,
but are not limited to, β-alanine, L-α-amino butyric acid, L-γ-amino-butyric acid, L-α-amino
isobutyric acid, L-ε-amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L-glutamic
acid, cysteine (acetamindomethyl), N-ε-Boc-N-α-CBZ-L-lysine, N-ε-Boc-N-α-Fmoc-L-lysine,
L-methionine sulfone, L-norleucine, L-norvaline, N-α-Boc-N-5-CBZ-L-omithine, N-δ-Boc-N-

α-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, Boc-L-thioproline.
(Blondelle, et al. (1994) Antimlcrob. Agents Chemother. 38(10): 2280-6; Pinilla, et al. (1995)
Biopolymers. 37(3): 221-40).
[0089] Sometimes, the binding agents can be small molecules that bind, interact, or
associate with a protein. Such a small molecule can be an organic molecule that is capable of
penetrating the lipid bilayer of a cell. Small molecules include, but are not limited to, toxins,
chelating agents, metals, and metalloid compounds. Small molecules can be attached or
conjugated to a targeting agent so as to specifically guide the small molecule to a particular
cell.
[0090] In some embodiments, a binding agent is a nucleic acid sequence, which can be a
full-length sequence, fragments of full-length sequences or synthesized oligonucleotides that
bind under physiological conditions to a protein such as a transcription factor. "Nucleic acid"
refers to a polymer comprising two or more nucleotides and includes single-, double-, and
triple-stranded polymers. "Nucleotide" refers to both naturally occurring and non-naturally
occurring compounds and comprises a heterocyclic base, a sugar, and a linking group, such as a
phosphate ester. For example, structural groups may be added to the ribosyl or deoxyribosyl
unit of the nucleotide, such as a methyl or allyl group at the 2'-O position or a fluoro group that
substitutes for the 2'-O group. The linking group, such as a phosphodiester, of the nucleic acid
may be substituted or modified, for example with methyl phosphonates or O-methyl
phosphates. Bases and sugars can also be modified, as is known in the art. "Nucleic acid," for
the purposes of this disclosure, also includes "peptide nucleic acids" in which native or
modified nucleic acid bases are attached to a polyamide backbone.

[0091] The binding agents of the present invention can be conjugated to a detectable label.
According to the invention, a "detectable label" is a moiety that can be sensed. In some
embodiments, a detectable label is operably linked to a binding agent. By "operably linked," it
is meant that the detectable label is attached to the binding agent by either a covalent or non-
covalent (e.g., ionic) bond. Methods for creating covalent bonds are known (see general
protocols in, e.g., Wong, S. S. (1991) Chemistry of Protein Conjugation and Cross-Linking,
CRC Press; Burkhart et al. (1999) The Chemistry and Application of Amino Crosslinking
Agents or Aminoplasts, John Wiley & Sons Inc.).
[0092] In accordance with the invention, a detectably labeled binding agent includes a
binding agent that is conjugated to a detectable moiety. Another detectably labeled binding
agent of the invention is a fusion protein, where one partner is the binding agent and the other
partner is a detectably label. Yet a further non-limiting example of a detectably labeled binding
agent is a first fusion protein comprising a binding agent and a first moiety with high affinity a
second moiety, and a second fusion protein comprising a second moiety and a detectable label.
For example, a binding agent that specifically binds to a protein is operably linked to a
streptavidin moiety. A second fusion protein comprising a biotin moiety operably linked to a
fluorescein moiety is added to the binding agent-streptavidin fusion protein, where the
combination of the second fusion protein to the binding agent-streptavidin fusion protein
results in a detectably labeled binding agent (i.e., a binding agent operably linked to a
detectable label). In particular embodiments, the detectable label is detectable by a medical
imaging device or system. For example, where the medical imaging system is an X-ray
machine, the detectable label that can be detected by the X-ray machine is a radioactive label
(e.g., 32P). Note that a binding agent need not be directly conjugated to the detectable moiety.

For example, a binding agent (e.g., an antibody) that is itself specifically bound to by a
secondary detectable binding agent (e.g., a FITC labeled goat anti-mouse secondary antibody)
is operably linked to a detectable moiety (i.e., the FITC moiety).
[0093] Detectable labels can be, without limitation, fluorophores (e.g., fluorescein (FITC),
phycoerythrin, rhodamine), chemical dyes, or compounds that are radioactive,
chemoluminescent, electrochemiluminescent, magnetic, paramagnetic, promagnetic, or
enzymes that yield a product that may be colored, chemoluminescent, or magnetic. The signal
is detectable by any suitable means, including spectroscopic, photochemical, biochemical,
immunochemical, electrical, optical or chemical means. In certain cases, the signal is
detectable by two or more means. In certain embodiments, protein labels include fluorescent
dyes, radiolabels, electrochemiluminescent, and chemiluminescent labels.
[0094] For example, amino acids of binding agents may be conjugated to Cy5/ Cy3
fluorescent dyes. These dyes are frequently used in the art (see, e.g., Linder et al. (2002)
Electrophoresis. 23(5): 740-9). The fluorescent labels can be selected from a variety of
structural classes, including the non-limiting examples such as 1- and 2-aminonaphthalene,
p,p'diaminostilbenes, pyrenes, quaternary phenanthridine salts, 9-aminoacridines, p,p'-
diaminobenzophenone imines, anthracenes, oxacarbocyanine, marocyanine, 3-aminoequilenin,
perylene, bisbenzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol, bis-3-
aminopridinium salts, hellebrigenin, tetracycline, sterophenol, benzimidazolyl phenylamine, 2-
oxo-3-chromen, indole, xanthen, 7-hydroxycoumarin, phenoxazine, salicylate, strophanthidin,
porphyrins, triarylmethanes, flavin, xanthene dyes (e.g., fluorescein and rhodamine dyes);
cyanine dyes; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes and fluorescent proteins (e.g.,
green fluorescent protein, phycobiliprotein).

[0095] Other useful dyes are chemiluminescent dyes and can include, without limitation,
biotin conjugated amino acids. In particular embodiments, electrochemiluminescent probes are
conjugated to binding agents. As used herein, "electrochemiluminescence" is a
chemiluminescent reaction that occurs subsequently to an electrochemical reaction.
Electrochemiluminescent probes include, but are not limited to, luminol, acridan ester,
ruthenium, ruthenium chelate, and ruthenium tribipyridine, NHS ester.
Electrochemiluminescent probes can be obtained commercially from, e.g., BioVeris Corp.
(Gaithersburg, MD).
1.3 Analytical Assays
[0096] Aspects of the present invention also utilize assays of a protein's quality. These
assays can be utilized during high-throughput titer screening of the protein-of-interest. Protein
quality can also be determined during cell culture process development. As part of protein
quality, protein structure includes the primary, secondary, tertiary, and quaternary structure of a
protein as well as post-translational modifications such as glycosylation, lipidation, and
phosphorylation. In addition, the size, shape, and charge of the protein affect the quality of the
protein. The physical structure of a protein has a significant effect on the ability of a protein to
perform its normal functions. In the context of enzymatic reactions, protein structure is vitally
important for full enzymatic quality. In the context of antibodies or fragments thereof, the size,
shape, surface charge, glycosylation, and phosphorylation of amino acids of the antibody has a
significant effect on the antibody's epitope specificity.
[0097] In some embodiments, NMR, Matrix assisted laser desorption/time-of-flight
("MALDI-TOF") analysis, and circular dichroism are used to determine the physical structure
of a protein (see, e.g., U.S. Patent Nos. 6,930,305; 7,005,272; and 7,029,872). Such techniques

provide a detailed analysis of a protein's overall physical structure. These techniques are well
known in the art.
[0098] In particular embodiments, size exclusion chromatography is used to determine the
size of the protein of interest (see, e.g., Brooks et al. (2000) Proc. Natl. Acad. Sci USA. 97(13):
7064-7067). In addition, cation exchange chromatography can be used to determine the charge
of a protein (see, e.g., Zhang and Glatz (1999) Biotechnol. Prog. 15(1): 12-18). Other
techniques that can be utilized to identify the structure of a protein-of-interest include, but axe
not limited to, reverse phase HPLC, capillary electrophoresis SDS, capillary zone
electrophoresis, and high pH anionic exchange HPLC. These techniques can be practiced
using procedures that are well known in the art.
[0099] Other structural assays include the sialic acid assay and the lectin assay. These
assays identify the level of glycosylation found on the surface of a protein, which informs on
the quality of the protein. Sialic acid assays have been used to determine the extent of
carbohydrates present in a sample, and these techniques are known in the art (see, e.g., U.S.
Patent Nos. 5,807,553 and 5,855,901). Lectin based assays also detect the presence of
carbohydrate in a sample, but through a mechanism of a protein-carbohydrate interaction (see,
e.g., U.S. Patent No. 5,633,148). Lectin assays have been used extensively in the art for
carbohydrate binding, and are described, for example, in U.S. Patent No. 6,331,319.
[0100] In addition to determining the physical structure of a protein, the ability of a protein
to perform certain functions can be assayed (see, e.g., U.S. Patent No. 7,029,862). Also, the
binding function of a protein or polypeptide (e.g., encoded by hybridizing nucleic acid) can be
detected in binding or binding inhibition assays, using membrane fractions containing receptor
or cells expressing receptor, for example (see e.g., Van Riper et al. (1993) J. Exp. Med., 177:

851 856; Sledziewski et al., U.S. Pat. No. 5,284,746). Thus, the ability of the encoded protein
or polypeptide to bind a ligand, an inhibitor and/or promoter, can be assessed. The antigenic
properties of proteins or polypeptides encoded by nucleic acids of the present invention can be
determined by immunological methods employing antibodies that bind to a protein, such as
immunoblotting, immunoprecipitation and immunoassay (e.g., radioimmunoassay, ELISA).
[0101] The signaling function of a protein or polypeptide (e.g., encoded by hybridizing
nucleic acid) can be detected by enzymatic assays. The stimulatory function of a protein or
polypeptide (e.g., encoded by hybridizing nucleic acid) can be detected by standard assays for
chemotaxis or mediator release, using cells expressing the protein or polypeptide (e.g., assays
which monitor chemotaxis, exocytosis (e.g., degranulation of enzymes, such as esterases (e.g.,
serine esterases), perforin, granzymes) or mediator release (e.g., histamine, leukotriene) in
response to a ligand or a promoter (see e.g., Taub et al. (1995) J. Immunol., 155: 3877-3888;
Baggliolini, M. and C. A. Dahinden (1994) Immunology Today, 15: 127-133 and references
cited therein). Functions characteristic of a protein receptor can also be assessed by other
suitable methods.
[0102] To demonstrate the methods according to the invention, screening methods as
described above were performed on various cell lines for the purpose of identifying those cell
lines that produced both a sufficient quantity of protein and sufficient quality of protein.
EXAMPLES
[0103] Those skilled in the art will recognize, or be able to ascertain, using no more than
routine experimentation, numerous equivalents to the specific substances and procedures

described herein. Such equivalents are intended to be encompassed in the scope of the claims
that follow the examples below.
EXAMPLE 1
Human Fc Protein Assay High-throughput Screening
1. Preparation of Anti-Ap Standards
[0104] A Standard Curve Buffer (SCB) was prepared by mixing 20 ul of Media R5CD1,
and 24 ml Assay Buffer (PBS w/ 0.05 % Tween 20 and 1 % bovine serum albumin) in 16 assay
plates (Corning/Costar, Corning, NY). A 0.3 mg/ml intermediate standard was prepared using
6 ul of 32.5 mg/ml reference standard and 644 ul SCB (prepared fresh each time). One ug/ml
standard was placed into a well designated Al and into an additional well designated HI in a
2ml deep-well plate. Well Al contained 6 ul of 0.3 mg/ml intermediate standard and 1794 ul
of SCB in 16 assay plates. This process was repeated for well H1. Serial dilutions were
prepared by taking 900 ul of solution in the wells and adding 900 ul of SCB to each of the 16
assay plates. The dilutions were distributed 50 ul per standard well.
2. Preparation of Controls
[0105] Controls were prepared to produce a concentration of 120 ug/ml intermediate
control. Briefly, 6 ul of 32.5 mg/ml reference standard and 1619 ul of Media R5CD1 were
mixed. Dilutions of 1:40 were prepared by mixing 5 ul of 120ug/ml intermediate control and
195 ul of Assay Buffer, while dilutions of 1:400 were prepared by mixing 20 ul of 1:40 diluted
control and 180 ul of Assay Buffer. Dilutions of 1:1200 dilution were prepared by taking 80 ul

of 1:400 diluted control and mixing in 160 ul Assay Buffer per every two assay plates. The
1.1200 dilutions were distributed in SO ul amounts per 0.1 control well.
[0106] In addition, controls containing 0.01 μg/ml of control were prepared by mixing 150
ul of 120 ug/ml intermediate control in (prepared as above) in 1350 ul of Media. The 1:40
dilution was prepared by mixing 5 ul of 12ug/ml intermediate control in 195 ul of Assay
Buffer. The 1:400 dilution was prepared by mixing 20 ul of 1:40 diluted control in 180 ul of
Assay Buffer and the 1:1200 dilution was prepared by mixing 80 ul of 1:400 diluted control in
160 ul Assay Buffer /2 assay plates. The controls were distributed in 50 ul aliquots of 1:1200
diluted control per 0.01 control well.
2. Preparation of ORI-Tagged F(ab')2 Fragment
[0107] Preparation of ORI-tagged Anti-Fc F(ab')2 fragment (Jackson ImmunoResearch
Laboratories, Inc., West Grove, PA) was accomplished using the following protocol. Briefly,
50 μl DMSO was added to one vial of ORITAG NHS ester (BioVeris, Gaithersburg, MD). The
mixture was vortexed at the maximum setting until the ORITAG in the bottom of the vial
dissolved. Then, 1638 μl of affiniPure F(ab')2 Fragment Goat Anti-Human IgG antibody was
added to 50 μl of dissolved ORITAG NHS ester to 1638 ul. The mixture was vortexed and
incubated at room temperature for 60 minutes in a dark wrap. The vial was rotated on a rocker
during the incubation.
[0108] The reaction was stopped by adding 20 μl of 2M glycine, and the tube was wrapped
with foil and incubated for 10 minutes at room temperature. During incubation, two PDIO
(Pharmacia, Piscataway, NJ) columns with PBS were equilibrated with 0.1% NaN3 and used
according to manufacturer's protocol. The reaction tubes were centrifuged for 5 seconds to

collect all of the volume in the tube. Then, 8S4 ul of the reaction was added to each PD10
columns. The samples were loaded to the columns and eventually 8 tubes of 0.5 ml aliquots
were collected from each column.
[0109] The protein concentration was determined by BCA Protein Assay kit. In addition,
the un-labeled leftover antibody from the second step was used as a standard to determine
protein concentration. The absorbance of the protein samples was measured at 455 nra.
[0110] Fractions with appropriate protein concentrations and good ORI-TAG: Protein ratio
were pooled. Bovine serum albumin was added to the final vial to make 1 % BSA solutions.
Vials were stored at 40 °C.
3. Cell Line Sample Preparation and Reaction
[0111] A 1:40 dilution was prepared by mixing 5 ul of a sample from a cell line generating
GPlbα, IL13R, anti-CD22 antibody, anti-Lewis Y antibody, anti-Aβ antibody, or TNFR fusion
protein and 195 ul of Assay Buffer, while a 1:400 dilution of sample was prepared by mixing
20 ul of 1:40 diluted sample and 180 ul of Assay Buffer. Finally, a 1:1200 dilution was
prepared by taking 70 ul of 1:400 diluted samples and mixing the samples with 140 ul Assay
Buffer. This final dilution was distributed in 50 ul to each sample well. The 1:400 were also
distributed to sample wells in another assay plate.
[0112] The tagged F(ab')2 fragment was distributed in aliquots of 5350 μl comprising 6 ul
of F(ab')2 fragment in 5344 ul Buffer to each assay plate. There was 50 ul of solution per well.
[0113] Protein A beads were obtained from Dynal Biotech (Carlsbad, CA). Beads were
distributed in 30 ul quantities in 5 ml of Assay buffer per plate. The solutions was distributed
in 50 ul volumes per well.

[0114] All reagents and samples were distributed to the plates as follows: The load
standard, control, and samples were loaded into the wells first. Then the anti-Fc ORI-tagged
F(ab')2 fragment was loaded. Finally, the Protein A beads were loaded.
[0115] The mixture was incubated for 2 hours at room temperature with mixing, and read
on an M8 or M384 analyzer with method "FcHuman150."
4. Data Analysis
[0116] The Standard Acceptance Guidelines used for the experiments were at least 8 out of
10 standards the CV of the readings between standard point replicates should be about 20%. In
addition, the Control Acceptance Guidelines had to be within 80 to 120 %. Also, the Sample
Acceptance Guidelines required a CV of the readings between sample replicates to be about
20%. Only the readings that fell within these ranges were taken into consideration.
5. Results
[0117] " The labeling of the F(ab')2 fragment with ruthenium directed against PSGL showed
significantly higher signal to noise ratios (Fig. 2). The data for 0.1 μg/ ml and 0.4 μg/ ml of
F(ab')2 fragments were normalized against noise and placed onto the bar graph. The Jackson 1,
Jackson 2, Rockland 1, and Southern Biotech F(ab')2 fragments had increased signal to noise
ratios when labeled with ruthenium (Fig. 2). These experiments were confirmed using F(ab')2
fragments directed against anti-CD22 (Fig. 3).
[0118] These results were further detailed in experiments utilizing F(ab')2 fragments
directed against the Fc fusion proteins GPlbα, IL13 receptor, and TNFR fusion protein (Fig.
4). The plot shows that as the concentration of the target fusion proteins increased, the

detection of the proteins with the F(ab')2 fragments increased (Fig. 4). Similar results were
also obtained using anti-GDF8, anti-CD22, and anti-Lewis Y antibodies as the target rather
than the Fc fusion proteins (Fig. 5).
[0119] Using the labeled F(ab')2 fragments to identify the quantity of protein in a sample,
the titer screening methodology was tested against standard column procedures such as HPLC.
The results showed that the titer screening methodology was much faster rates than HPLC
chromatography to determine the titer of the proteins (Fig. 6). The time required to obtain the
protein quantity readings was more than 10 times greater using HPLC as compared to the high-
throughput titer screening when up to over 700 samples were analyzed (Fig. 6).
[0120] In addition to the high-throughput screening being faster than standard column
procedures, it is as accurate in determining protein quantity (Figs. 7,9,10, and 11). When
HPLC and the titer screening procedures detailed above were compared, near identical titer
quantities were identified for anti-Lewis Y protein, PSGL, anti-Aβ, and TNFR fusion protein
(Figs. 7,9,10, and 11). Accordingly, the titer screening assay detailed herein was faster and
had a similar efficiency as the standard column techniques at determining protein quantity.
[0121] The high-throughput screen utilized above was able to identify cell lines that were
expressers of the appropriate quantity of antibody (Fig. S). As shown in Figure 8, the high-
throughput titer screening allowed for the identification of the highest producers of the
antibody-of-interest, which is indicated by the increasing titer (μg/ ml) per clone. In these
experiments, the top titer clone is numbered 1, the second highest titer clone is numbered two,
and so on. Therefore, the top clones were quickly identified by the assay.

EXAMPLE 2
High-throughput Purification and Identification of Proper Cell Culture Conditions
1. Manual Purification of Proteins Using Centrifugation
[0122] The high-throughput titer screening procedure of Example 1 can be linked to the
high-throughput purification procedure detailed below to improve the potential development of
cell culture development.
[0123] The thin resin came in 20% Ethanol. Additional 20% Ethanol solution was added to
make up 50% of the settled volume. The solution was mixed thoroughly and dispensed in 200
uL aliquots per well of a filterplate (Whatman 7700-2804, long drip, 25 tun, 96 well x 800 uL,
Whatman LabSciences, Orange, NJ). The filterplates were stacked on top of empty
microplates (Corning/Costar, Corning, NY), and centrifuged (Sorvall Legend RT) for 3 min at
700 rpm (approximately equals 104 G ) to remove the 20% Ethanol. Then, 200 uL per well of
RODI water was added, and the plates were centrifuged for 3 min with empty microplates
underneath. This process was repeated twice.
[0124] Wash buffer was added at 200 uL aliquots per well. The plates were centrifuged for
3 min with empty microplates underneath. This process was repeated two more times. When
all samples were at least 170 ug/mL, the minimal dilution was made to make the titers of the
same set of samples to be approximately the same range (+/- 20%). The samples were diluted
to be close to the lowest concentration. Any samples under 170 ug/mL were loaded more than
once. When multiple loading was required, the samples were diluted only to ensure that the
total mass of protein loaded was in the same range (+/- 20%). When multiple loading was

performed, the samples that required were added first and in the empty wells 200 uL of wash
buffer was added. The filterplates were centrifuged as needed.
[0125] All samples were loaded together with reference material spikes and media blanks
as usual. The samples were loaded onto the Protein A resin (Protein A Mab Select, Amersham
Biosciences) at a load volume of 500 uL. A spiking standard was prepared in media with the
concentration close to the whole set of test samples. The spiking material and media blank
were loaded at 500ul/well.
[0126] The resin was resuspended with sample by mixing with multi-channel pipette. The
resin and sample were incubated for 5-10 minutes at room temperature. The mixture was then
centrifuged at 700 rpm for 3 min., and the sample flow-through was collected in a deep well
microplate (Whatman 7710-5750, Whatman LabSciences, Orange, NJ). Then, 200 uL per well
of wash buffer (5 mM Tris, 20, 50 or 100 mM NaCl, pH 7.5) was added and the mixture was
centrifuged for 3 min with empty microplates underneath. Wash buffer was added at 200 uL
per well. The samples were centrifuged for 3 min with empty microplates underneath. Then,
4uL Tris (2.0 M Tris, pH 8.5, or 1.0 M Tris pH 8.5) was added to the mixture for neutralization
to the UV collection plate (Coming/Costar, Coming, NY) before performing elution. Elution
buffer (50 mM Glycine, 20 or 50 or 100 mM NaCl, pH 2.5 or 3.0) was added at 200 uL
volumes per well to filter plate. The resin and the elution buffer were mixed with a multi-
channel pipette. The filter plate was then centrifuged for 3 min with the collection plate
underneath. Plates were read at A280 on a Spectra Max plate reader (Molecular Devices).

2. Protein Quality Determinations Using Column Chromatography
[0127] For CEX assay: The elution was transferred at 50 uL aliquots into an Agilent plate
containing 100 uL of CEX mobile phase A. The solution was mixed. Column purification
procedures were performed using standard procedures.
[01281 For SEC assay: The elution was transferred atl 30 uL volumes to an Agilent plate.
The solution was mixed. Column purification procedures were performed using standard
procedures.
[0129] For HIC and Sialic acid assay: Four microliters of 2M Tris was added to the UV
plate and eluted directly into UV plate. The solution was mixed and read at A280 on a Spectra
Max plate reader.
3. Results
[0130] High-throughput purification of samples using the above techniques allowed for
rapid purification of up to 96 samples per plate. The purification techniques produced rapid
purification of samples without a decrease in the level of purification (Figs. 12 and 13). As
determined by the amount of high molecular weight protein present in each sample, the high-
throughput purification techniques performed as well as other techniques (Figs. 12 and 13). In
particular, when comparing the high-throughput purification to standard Protein A purification
(Fig. 12 and 13).
[0131] Furthermore, the amount of high molecular weight protein was used to determine
the quality of protein generated by cells grown in different cell culture conditions (Fig. 14). As
shown in Figure 14, the various media tested showed different quantities of high molecular
weight protein after purification that appeared to be dependent on the media. In particular,

certain media conditions showed statistically significant improvements in the amount of high
molecular weight protein as compared to the other media (Fig. 14, large arrows). Therefore,
the cell culture process development procedure utilized above identified media that showed
improved growth characteristics for downstream purification.
EQUIVALENTS
[0132] Those skilled in the art will recognize, or be able to ascertain, using no more than
routine experimentation, numerous equivalents to the specific compositions and procedures
described herein. Such equivalents are considered to be within the scope of this invention, and
are covered by the following claims.

CLAIMS
1. 1. A method of high-throughput screening of cell lines for protein expression,
comprising:
a) screening samples obtained from cell lines to determine a level of expression for
a protein-of-interest in each cell line by contacting the samples with a first binding
agent;
b) contacting the protein-of-interest with a second binding agent operably linked to
a detectable label;
c) determining the level of expression for the protein-of-interest;
d) determining an appropriate quality for the protein-of-interest;
e) selecting a cell line for high-throughput protein expression of the protein-of-
interest;
wherein a cell line is selected if the cell line produces a desired level of expression
and an appropriate quality for the protein-of-interest.
2. The method of claim 1, wherein the protein-of-interest is selected from the group
consisting of antibodies, ligands, receptors, subunits of proteins, fragments of proteins, fusion
proteins, recombinant proteins, and fragments of the same.
3. The method of claim 2, wherein the protein-of-interest is an antibody, a recombinant
antibody, or a F(ab')2 fragment
4. The method of claim 1, wherein the first binding agent is selected from the group
consisting of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant
proteins, and fragments of the same.

5. The method of claim 4, wherein the first binding agent is Protein A or streptavidin.
6. The method of claim 4, wherein the first binding agent can be attached to a solid
support selected from the group consisting of beads, plates, and microarray chips.
7. The method of claim 6, wherein the solid support comprises cellulose, sepharose,
polyacrylamide, glass, or polystyrene.
8. The method of claim 1, wherein an appropriate quality for the protein-of-interest is
selected from the group consisting of charge, size, enzymatic activity, antibody-epitope
interaction, nucleic acid binding, carbohydrate content, secondary structure, tertiary structure,
and binding activity.
9. The method of claim 1, wherein the second binding agent is selected from the group
consisting of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant
proteins, and fragments of the same.
10. The method of claim 9, wherein the second binding agent is an antibody or fragments of
the same.

11. The method of claim 10, the antibody is a F(ab')2 fragment,
12. The method of claim 11, wherein the F(ab')2 fragment specifically binds to the Fc
portion of an antibody.
13. The method of claim 1, wherein the ruthenium labeled second binding agent is labeled
with a second detectable label selected from the group consisting of fluorophores, chemical
dyes, radioactive binding agents, chemiluminescent binding agents, electrochemiluminescent
agents, magnetic binding agents, paramagnetic binding agents, promagnetic binding agents,
enzymes that yield a colored product, enzymes that yield a chemiluminescent product, and
enzymes that yield a magnetic product

14. The method of claim 12, wherein the F(aV)2 fragment is operably linked to two or
more ruthenium labels.
15. The method of claim 1, wherein the samples are contacted by a third binding agent
attached to a resin.
16. The method of claim 15, wherein the third binding agent is selected from the group
consisting of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant
proteins, and fragments of the same.
17. The method of claim 16, wherein the third binding agent is Protein A or streptavidin.
18. The method of claim 17, wherein the third binding agent is attached to a solid support.
19. The method of claim 1, wherein the resin is isolated from the mixture and the expressed
protein-of-interest is eluted from the third binding agent.
20. The method of claim 19, wherein the protein-of-interest is eluted by a method selected
from the group consisting of vacuum elution and gravity flow.
21. The method of claim 1, wherein the cell lines are screened using an automated
workstation.
22. A method of high-throughput screening of cell lines for protein expression and
production, comprising:

a) contacting a solid support with a sample isolated from a cell line, the solid
support having a first binding agent attached to its surface, the first binding agent
being capable of binding to a protein-of-interest;
b) contacting the sample with a second binding agent that binds to the protein-of-
interest, the second binding agent being operably linked to a detectable label;

c) determining the level of expression of the protein-of-interest by detecting the
label operably linked to the second binding agent that is bound to the protein-of-
interest; and
d) comparing the level of expression of the protein-of-interest in each cell line to the
average level of expression of the protein-of-interest and selecting the cell line
based on the comparison,
wherein a cell line is selected for protein production if the level of expression of the
protein-of-interest in the cell line is either greater than or less than the average level of
expression of the protein-of-interest in all cell lines.
23. The method of claim 22 further comprising isolating supernatants from the selected cell
lines, and contacting, the supernatants with a reagent that binds to the protein-of-interest.
24. The method of claim 23, wherein the reagent is attached to a solid support.
25. The method of claim 24, wherein the solid support is a multiwell plate.
26. The method of claim 23, wherein the bound protein-of-interest is eluted from the
reagent and assayed for appropriate quality.
27. The method of claim 26, wherein the cell line is selected for protein expression if the
cell line is selected in step e), and the expressed protein-of-interest has the appropriate quality.
28. The method of claim 22, wherein the protein-of-interest is selected from the group
consisting of antibodies, ligands, receptors, subunits of proteins, fragments of proteins, fusion
proteins, recombinant proteins, and fragments of the same.
29. The method of claim 28, wherein the protein-of-interest is an antibody, a recombinant
antibody, or a F(ab')2 fragment

30. The method of claim 22, wherein the first binding agent is selected from the group
consisting of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant
proteins, and fragments of the same.
31. The method of claim 30, wherein the first binding agent is Protein A or streptavidin.
32. The method of claim 22, wherein the binding agents can be attached to a solid support
selected from the group consisting of beads, plates, and microarray chips.
33. The method of claim 24, wherein the solid support comprises cellulose, sepharose,
polyacrylamide, glass, or polystyrene.
34. The method of claim 22, wherein the second binding agent is selected from the group
consisting of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant
proteins, and fragments of the same.
35. The method of claim 34, wherein the second binding agent is an antibody or fragments
of the same.
36. The method of claim 35, wherein the antibody is a F(ab')2 fragment.
37. The method of claim 35, wherein the antibody is an F(ab')2 fragment that specifically
binds to the Fc portion of an antibody.
38. The method of claim 22, wherein the detectable label is selected from the group
consisting of fluorophores, chemical dyes, radioactive binding agents, chemiluminescent
binding agents, electrochemihiminescent agents, magnetic binding agents, paramagnetic
binding agents, promagnetic binding agents, enzymes that yield a colored product, enzymes
that yield a chemiluminescent product, and enzymes that yield a magnetic product.
39. The method of claim 38, wherein the detectable label is ruthenium.

40. The method of claim 22, wherein a reagent comprises a resin that has a third binding
agent attached to it, the third binding agent being capable of binding to the protein-of-interest.
41. The method of claim 40, wherein the third binding agent is selected from the group
consisting of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant
proteins, and fragments of the same.
42. The method of claim 41, wherein the third binding agent is Protein A or streptavidin.
43. The method of claim 40, wherein the resin is isolated, and the protein-of-interest is
eluted from the third binding agent
44. The method of claim 43, wherein the protein-of-interest is eluted from the third binding
agent using a method selected from the group consisting of vacuum elution and gravity flow.
45. The method of claim 22, wherein the screening of the incubated cell lines utilizes an
automated workstation.
46. A method of cell culture process development, comprising:

a) incubating each cell line in a different cell culture condition;
b) contacting each cell line sample with first binding agent attached to a solid
support, the first binding agent binding to a protein-of-interest in the cell line
samples;

c) contacting the protein-of-interest bound by the first binding agent with a second
binding agent, which is operably linked to a detectable label;
d) determining the level of expression for the protein-of-interest by detecting the
label operably linked to the second binding agent that is bound to the protein-of-
interest; and

e) selecting the cell line based on the detected level of expression of the protein-of-
interest,
wherein a cell line is selected if the level of expression of the protein-of-interest in
the cell line is greater than or less than the average level of expression of the protein-of-interest
in all of the cell lines.
47. The method of claim 46 further comprising isolating supernatants from the selected cell
lines, and contacting the supernatants with a reagent that binds to the protein-of-interest.
48. The method of claim 47, wherein the reagent is attached to a solid support.
49. The method of claim 48, wherein the solid support is a multiwell plate.
50. The method of claim 47, wherein the bound protein-of-interest is eluted from the
reagent and assayed for appropriate quality.
51. The method of claim 50, wherein the cell line is selected for protein expression if the
cell line is selected in step e), and the expressed protein-of-interest has the appropriate quality.
52. The method of claim 46, wherein the protein-of-interest is selected from the group
consisting of antibodies, ligands, receptors, subunits of proteins, fragments of proteins, fusion
proteins, recombinant proteins, and fragments of the same.
53. The method of claim 52, wherein the protein-of-interest is an antibody, a recombinant
antibody, or a F(ab')2 fragment.
54. The method of claim 46, wherein the first binding agent is selected from the group
consisting of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant
proteins, and fragments of the same.
55. The method of claim 54, wherein the first binding agent is Protein A or streptavidin.

56. The method of claim 46, wherein the binding agents can be attached to a solid support
selected from the group consisting of beads, plates, and microarray chips.
57. The method of claim 48, wherein the solid support comprises cellulose, sepharose,
polyacrylamide, glass, or polystyrene.
58. The method of claim 46, wherein the second binding agent is selected from the group
consisting of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant
proteins, and fragments of the same.
59. The method of claim 58, wherein the second binding agent is an antibody or fragments
of the same.
60. The method of claim 59, wherein the antibody is a F(ab')2 fragment.
61. The method of claim 59, wherein the antibody is an F(ab')2 fragment that specifically
binds to the Fc portion of an antibody.
62. The method of claim 46, wherein the detectable label is selected from the group
consisting of fluorophores, chemical dyes, radioactive binding agents, chemiluminescent
binding agents, electrochemiluminescent agents, magnetic binding agents, paramagnetic
binding agents, promagnetic binding agents, enzymes that yield a colored product, enzymes
that yield a chemiluminescent product, and enzymes that yield a magnetic product.
63. The method of claim 62, wherein the detectable label is ruthenium.
64. The method of claim 46, wherein a reagent comprises a resin that has a third binding
agent attached to it, the third binding agent being capable of binding to the protein-of-interest
65. The method of claim 64, wherein the third binding agent is selected from the group
consisting of antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant
proteins, and fragments of the same.

66. The method of claim 65, wherein the third binding agent is Protein A or streptavidin.
67. The method of claim 64, wherein the resin is isolated, and the protein-of-interest is
eluted from the third binding agent.
68. The method of claim 67, wherein the protein-of-interest is eluted from the third binding
agent using a method selected from the group consisting of vacuum elution and gravity flow.
69. The method of claim 46, wherein the screening of the incubated cell lines utilizes an
automated workstation.

Disclosed are methods for high-throughput screening of cell lines for use in protein expression in certain pharmaceutical,
drug development, and biotechnological processes such that high productivity cell lines are identified for their ability to
produce both desired levels of protein expression and appropriate quality of a protein-of- interest.