U.S. patent application number 17/544796 was filed with the patent office on 2022-09-29 for method to improve virus filtration capacity.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Amit Mehta.
Application Number | 20220306726 17/544796 |
Document ID | / |
Family ID | 1000006394592 |
Filed Date | 2022-09-29 |
United States Patent
Application |
20220306726 |
Kind Code |
A1 |
Mehta; Amit |
September 29, 2022 |
METHOD TO IMPROVE VIRUS FILTRATION CAPACITY
Abstract
The present invention relates to the field of protein
purification. In particular, the invention concerns methods for
increasing the filtration capacity of virus filters, by combined
use of endotoxin removal and cation-exchange media in the
prefiltration process.
Inventors: |
Mehta; Amit; (Fremont,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
1000006394592 |
Appl. No.: |
17/544796 |
Filed: |
December 7, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16848564 |
Apr 14, 2020 |
11225513 |
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17544796 |
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12806171 |
Aug 6, 2010 |
10662237 |
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16848564 |
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61231811 |
Aug 6, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/065 20130101;
C07K 1/36 20130101; C07K 1/18 20130101; C07K 1/34 20130101 |
International
Class: |
C07K 16/06 20060101
C07K016/06; C07K 1/36 20060101 C07K001/36; C07K 1/18 20060101
C07K001/18; C07K 1/34 20060101 C07K001/34 |
Claims
1. A method of improving the filtration capacity of a virus filter
during protein purification, comprising subjecting a composition
comprising a protein to be purified to a cation exchange step and
an endotoxin removal step, simultaneously or in either order, prior
to passing through said virus filter.
2. The method of claim 1 wherein the pore size of the virus filter
is between about 15 and about 100 nm diameter.
3. The method of claim 2 wherein the pore size of the virus filter
is between about 15 and about 30 nm diameter.
4. The method of claim 3 wherein the pore size of the virus filter
is about 20 nm.
5. The method of claim 3 or claim 4 wherein the virus to be removed
is a parvovirus.
6. The method of claim 5 wherein the diameter of the parvovirus is
between about 18 and about 26 nm.
7. The method of claim 1 wherein the protein is an antibody or an
antibody fragment.
8. The method of claim 7 wherein the protein is a recombinant
antibody or antibody fragment.
9. The method of claim 8 wherein the recombinant antibody or
antibody fragment is produced in a mammalian host cell.
10. The method of claim 9 wherein the mammalian host cell is a
Chinese Hamster Ovary (CHO) cell.
11. The method of claim 1 wherein the composition comprising the
protein to be purified is first subjected to the cation exchange
step followed by the endotoxin removal step, prior to virus
filtration.
12. The method of claim 1 wherein the composition comprising the
protein to be purified is first subjected to the endotoxin removal
step followed by the cation exchange step, prior to virus
filtration.
13. The method of claim 1 wherein the composition comprising the
protein to be purified is subjected to the endotoxin removal step
and the cation exchange step simultaneously, prior to virus
filtration.
14. The method of claim 11 wherein said endotoxin removal step is
directly followed by virus filtration.
15. The method of claim 12 wherein said cation exchange step is
directly followed by virus filtration.
16. The method of claim 13 wherein said simultaneous endotoxin
removal and cation exchange step are directly followed by virus
filtration.
17. The method of claim 1 wherein virus filtration is performed at
a pH between about 4 and about 10.
18. The method of claim 1 wherein the protein concentration in said
composition is about 1-40 g/L.
19. The method claim 8 wherein said antibody is to one or more
antigens selected from the group consisting of HER1 (EGFR), HER2,
HER3, HER4, VEGF, CD20, CD22, CD11a, CD11b, CD11c, CD18, an ICAM,
VLA-4, VCAM, IL-17A and/or F, IgE, DR5, CD40, Apo2L/TRAIL, EGFL7,
NRP1, mitogen activated protein kinase (MAPK), and Factor D.
20. The method of claim 8 wherein the antibody is selected from the
group consisting of anti-estrogen receptor antibody,
anti-progesterone receptor antibody, anti-p53 antibody,
anti-cathepsin D antibody, anti-Bcl-2 antibody, anti-E-cadherin
antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9
antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody,
anti-CEA antibody, anti-retinoblastoma protein antibody, anti-ras
oncoprotein antibody, anti-Lewis X antibody, anti-Ki-67 antibody,
anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5
antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24
antibody, anti-CD10 antibody, anti-CD11c antibody, anti-CD13
antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19
antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31
antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35
antibody, anti-CD38 antibody, anti-CD41 antibody, anti-LCA/CD45
antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39
antibody, anti-CD100 antibody, anti-CD95/Fas antibody, anti-CD99
antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71
antibody, anti-c-myc antibody, anti-cytokeratins antibody,
anti-vimentins antibody, anti-HPV proteins antibody, anti-kappa
light chains antibody, anti-lambda light chains antibody,
anti-melanosomes antibody, anti-prostate specific antigen antibody,
anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin
antibody, anti-keratins antibody and anti-Tn-antigen antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/848,564, filed on Apr. 14, 2020, which is a
continuation of U.S. patent application Ser. No. 12/806,171, filed
on Aug. 6, 2010, now U.S. Pat. No. 10,662,237, which claims
priority under 35 U.S.C. Section 119(e) and the benefit of U.S.
Provisional Application No. 61/231,811, filed Aug. 6, 2009, the
entire disclosure of which is incorporated herein by reference in
its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention is from the field of protein
purification. In particular, the invention concerns methods for
increasing the filtration capacity of virus filters, by combined
use of endotoxin removal and cation-exchange media in the
prefiltration process.
Description of the Related Art
[0003] Mammalian cell lines have become the primary choice for
production of recombinant protein therapeutics due to their
capacity for proper protein folding and post translational
modification such as glycosylation (Chu and Robinson Current
Opinion in Biotechnology 12:180-187, 2001). However, these cell
lines are also known to contain retrovirus like particles (Lieber
et al. Science 182:56-59, 1973; Lubiniecki et al. Dev Biol Stand
70:187-191, 1989) and possess the risk for potential adventitious
virus contamination (Garnick, Dev Biol Stand. Basel: Karger
93:21-29, 1998). While the biopharmaceutical industry producing
recombinant protein drugs has a good safety record, there have been
past incidences of viral infection by blood and blood products
derived from plasma (Brown, Dev. Biol. Stand. 81, 1993; Thomas,
Lancet 343:1583-1584, 1994). To mitigate the risk of viral
contamination during recombinant protein production, downstream
purification processes are designed to include process steps that
remove endogenous and adventitious viruses. Adequate virus
clearance is obtained by a combination of several process steps
that provide either virus inactivation or virus removal from the
process feed stream. While viral inactivation is achieved using
techniques such as incubation at low pH, heat treatment, and
detergents, virus removal is typically performed using
chromatography and filtration (Curtis et al., Biotechnology and
Bioengineering 84(2):179-186, 2003).
[0004] Unlike chromatography media, which removes viruses based on
physicochemical properties such as net charge, virus filtration
removes viruses by size exclusion and is therefore considered a
more robust technique. So far usage of virus filtration during
downstream purification of biotherapeutics derived from mammalian
cell cultures has been limited to removal of retroviruses (80-100
nm diameter) due to lack of high throughput membranes with nominal
pore size less than 60 nm.
[0005] Recent advances in membrane technology have enabled
manufacturing of high throughput membranes with nominal pore size
of 20 nm. These virus filters are retentive to parvoviruses (18-26
nm diameter) and allow passage of proteins that are as large as 160
kD (.about.8 nm), e.g., monoclonal antibodies (mAbs).
[0006] The high selectivity and high throughput with parvovirus
filters is achieved by casting a thin retentive membrane layer on a
microporous substrate. The thin retentive layer while allows very
fine separation of proteins and viruses, it is also susceptible to
fouling by impurities in the process feedstream resulting in lower
filter capacity and flux. The fouling of the virus filters has been
attributed to contaminants such as protein aggregates and denatured
protein. Bohonak and Zydney (Bohonak and Zydney, Journal of
Membrane Science 254(1-2):71-79, 2005) showed that loss in filter
capacity could be due to cake formation or pore blockage. Other
recent reports (Bolton et al., Biotechnol. Appl. Biochem. 43:55-63,
2006; Levy et al., Filtration in the Biopharamaceutical Industry.
(Meltzer, T. H. and Jornitz, M. W., eds.) pp. 619-646, Marcel
Dekker, New York, 1998) have attributed the likely cause of filter
fouling to the adsorption of impurities to the pore walls. Several
publications (Bolton et al., Biotechnology and Applied Biochemistry
42:133-142, 2005; Hirasaki et al., Polymer Journal
26(11):1244-1256, 1994; Omar and Kempf, Transfusion
42(8):1005-1010, 2002) have also demonstrated that reduction in
filter capacity or plugging of pores can decrease viral retention
by few orders of magnitude, affecting the robustness of the unit
operation.
[0007] A lot of recent research has thus focused on identification
of pre-filters for removing the foulants from the process
feedstream to minimize virus filter fouling and ensuring high
capacity, high throughput and robust viral retention. Bolton et al.
(Bolton et al. 2006) performed a thorough study that involved
testing of several membranes as prefilters and demonstrated that it
was possible to increase capacity of normal flow parvovirus (NFP)
membranes by almost an order of magnitude by using
VIRESOLVE.TM.depth filter as a prefilter. Brown et al. (Brown et
al. 2008, Use of Charged Membranes to Identify Soluble Protein
Foulants in order to Facilitate Parvovirus Filtration. IBC's 20th
Antibody Development and Production, San Diego, Calif.) evaluated a
strong cation exchange membrane adsorber as a prefilter to
parvovirus retentive filter and showed that the capacity of virus
filter could be increased by several fold for eleven different mAb
streams. The authors hypothesized that the cation exchange membrane
adsorber removed large molecular weight (.about.600-1500 kD)
protein aggregates from the feedstream by competitive adsorption,
preventing the virus filter from plugging. U.S. Pat. No. 7,118,675
(Siwak et al.) describes a process that utilizes a charge-modified
membrane to remove protein aggregates from a protein solution to
prevent fouling of virus filter.
SUMMARY OF THE INVENTION
[0008] The present invention is based, at least in part, on the
experimental finding that fouling of parvovirus filters could be
due to impurities other than those mentioned in the literature and
more comprehensive prefiltration solutions are required to improve
the virus filtration capacity. Accordingly, the present invention
provides a novel prefiltration solution that performs significantly
better than the best prefiltration approach mentioned in the
literature (cation-exchange membrane adsorbers).
[0009] In one aspect, the invention concerns a method of improving
the filtration capacity of a virus filter during protein
purification, comprising subjecting a composition comprising a
protein to be purified to a cation exchange step and an endotoxin
removal step, in either order, prior to passing through said virus
filter.
[0010] In one embodiment, the pore size of the virus filter is
between about 15 and about 100 nm in diameter.
[0011] In another embodiment, the pore size of the virus filter is
between about 15 and about 30 nm in diameter.
[0012] In yet another embodiment, the pore size of the virus filter
is about 20 nm.
[0013] In a further embodiment, the virus to be removed is a
parvovirus.
[0014] In a still further embodiment, the diameter of the
parvovirus is between about 18 and about 26 nm.
[0015] In a different embodiment, the protein is an antibody or an
antibody fragment, such as an antibody produced by recombinant DNA
techniques, or a fragment thereof.
[0016] In another embodiment, the antibody is a therapeutic
antibody.
[0017] In yet another embodiment, the recombinant antibody or
antibody fragment is produced in a mammalian host cell, such as,
for example, a Chinese Hamster Ovary (CHO) cell.
[0018] In a further embodiment, the composition comprising the
protein to be purified is first subjected to a cation exchange step
followed by an endotoxin removal step, prior to virus
filtration.
[0019] In a still further embodiment, the composition comprising
the protein to be purified is first subjected to an endotoxin
removal step followed by a cation exchange step, prior to virus
filtration.
[0020] In another embodiment, the composition comprising the
protein to be purified is subjected to a cation exchange step and
endotoxin removal step simultaneously, prior to virus filtration,
by keeping the two media together in a single module.
[0021] In yet another embodiment, the endotoxin removal step is
directly followed by virus filtration.
[0022] In a further embodiment, the cation exchange step is
directly followed by virus filtration.
[0023] In a different embodiment, virus filtration is performed at
a pH between about 4 and about 10.
[0024] In another embodiment, the protein concentration in the
composition to be purified is about 1-40 g/L.
[0025] In yet another embodiment, the antibody to be purified is to
one or more antigens selected from the group consisting of HER1
(EGFR), HER2, HER3, HER4, VEGF, CD20, CD22, CD11a, CD11b, CD11c,
CD18, an ICAM, VLA-4, VCAM, IL-17A and/or F, IgE, DR5, CD40,
Apo2L/TRAIL, EGFL7, NRP1, mitogen activated protein kinase (MAPK),
and Factor D.
[0026] In a further embodiment, the antibody is selected from the
group consisting of anti-estrogen receptor antibody,
anti-progesterone receptor antibody, anti-p53 antibody,
anti-cathepsin D antibody, anti-Bcl-2 antibody, anti-E-cadherin
antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9
antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody,
anti-CEA antibody, anti-retinoblastoma protein antibody, anti-ras
oncoprotein antibody, anti-Lewis X antibody, anti-Ki-67 antibody,
anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5
antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24
antibody, anti-CD10 antibody, anti-CD11c antibody, anti-CD13
antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19
antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31
antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35
antibody, anti-CD38 antibody, anti-CD41 antibody, anti-LCA/CD45
antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39
antibody, anti-CD100 antibody, anti-CD95/Fas antibody, anti-CD99
antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71
antibody, anti-c-myc antibody, anti-cytokeratins antibody,
anti-vimentins antibody, anti-HPV proteins antibody, anti-kappa
light chains antibody, anti-lambda light chains antibody,
anti-melanosomes antibody, anti-prostate specific antigen antibody,
anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin
antibody, anti-keratins antibody and anti-Tn-antigen antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1: A schematic of the experimental setup used for virus
filtration studies.
[0028] FIG. 2: Effect of sterile and depth filter on the capacity
of Viresolve Pro parvovirus retentive filter. Experiments were
performed at pH 5.5 and conductivity of 8.5 mS/cm. mAb
concentration was approximately 13 g/L.
[0029] FIGS. 3 (a) and (b): Effect of cation-exchange and endotoxin
removal membrane adsorbers as prefilters on the capacity of
Viresolve Pro parvovirus filter. The data in 3 (a) and 3 (b) were
generated at pH 5.0 and 6.5 respectively with MAb1.
[0030] FIGS. 4 (a) and (b): Effect of a novel prefiltration train
containing both cation-exchange and endotoxin removal membrane
adsorbers on the capacity of Viresolve Pro parvovirus retentive
filter with MAb1. The data in 4 (a) and 4 (b) were generated at pH
5.0 and 6.5 respectively.
[0031] FIG. 5: Effect of a novel prefiltration train containing
both cation-exchange and endotoxin removal membrane adsorbers
compared to cation-exchange pre-filtration media on the capacity of
parvovirus retentive filter with MAb2.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
I. Definitions
[0032] By "protein" is meant a sequence of amino acids for which
the chain length is sufficient to produce the higher levels of
tertiary and/or quaternary structure. Thus, proteins are
distinguished from "peptides" which are also amino acid--based
molecules that do not have such structure. Typically, a protein for
use herein will have a molecular weight of at least about 15-20 kD,
preferably at least about 20 kD.
[0033] Examples of proteins encompassed within the definition
herein include mammalian proteins, such as, e.g., CD4, integrins
and their subunits, such as beta7, growth hormone, including human
growth hormone and bovine growth hormone; growth hormone releasing
factor; parathyroid hormone; thyroid stimulating hormone;
lipoproteins; .alpha.-1-antitrypsin; insulin A-chain; insulin
B-chain; proinsulin; follicle stimulating hormone; calcitonin;
luteinizing hormone; glucagon; clotting factors such as factor
VIIIC, factor IX, tissue factor, and von Willebrands factor;
anti-clotting factors such as Protein C; atrial natriuretic factor;
lung surfactant; a plasminogen activator, such as urokinase or
tissue-type plasminogen activator (t-PA, e.g., Activase.RTM.,
TNKase.RTM., Retevase.RTM.); bombazine; thrombin; tumor necrosis
factor-.alpha. and -.beta.; enkephalinase; RANTES (regulated on
activation normally T-cell expressed and secreted); human
macrophage inflammatory protein (MIP-1-.alpha.); serum albumin such
as human serum albumin; mullerian-inhibiting substance; mouse
gonadotropin-associated peptide; DNase; inhibin; activin; vascular
endothelial growth factor (VEGF); IgE, receptors for hormones or
growth factors; an integrin; protein A or D; rheumatoid factors; a
neurotrophic factor such as bone-derived neurotrophic factor
(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6),
or a nerve growth factor such as NGF-.beta.; platelet-derived
growth factor (PDGF); fibroblast growth factor such as aFGF and
bFGF; epidermal growth factor (EGF); transforming growth factor
(TGF) such as TGF-.alpha. and TGF-.beta., including TGF-.beta.1,
TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, or TGF-.beta.5; insulin-like
growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain
IGF-I); insulin-like growth factor binding proteins; other CD
proteins such as CD3, CD8, CD19 and CD20; erythropoietin (EPO);
thrombopoietin (TPO); osteoinductive factors; immunotoxins; a bone
morphogenetic protein (BMP); an interferon such as
interferon-.alpha., -.beta., and -.gamma.; colony stimulating
factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs),
e.g., IL-1 to IL-10; superoxide dismutase; T-cell receptors;
surface membrane proteins; decay accelerating factor (DAF); a viral
antigen such as, for example, a portion of the AIDS envelope;
transport proteins; homing receptors; addressins; regulatory
proteins; integrins such as CD11a, CD11b, CD11c, CD18, an ICAM,
VLA-4 and VCAM; a tumor associated antigen such as HER1 (EGFR),
HER2, HER3 or HER4 receptor; Apo2L/TRAIL, and fragments of any of
the above-listed polypeptides; as well as immunoadhesins and
antibodies binding to; and biologically active fragments or
variants of any of the above-listed proteins.
[0034] Specifically included within the definition of "protein," as
defined herein, are therapeutic antibodies and immunoadhesins,
including, without limitation, antibodies to one or more of the
following antigens: HER1 (EGFR), HER2, HER3, HER4, VEGF, CD20,
CD22, CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4, VCAM. IL-17A
and/or F, IgE, DR5, CD40, Apo2L/TRAIL, EGFL7, NRP1, mitogen
activated protein kinase (MAPK), and Factor D, and fragments
thereof.
[0035] Other exemplary antibodies include those selected from, and
without limitation, anti-estrogen receptor antibody,
anti-progesterone receptor antibody, anti-p53 antibody,
anti-cathepsin D antibody, anti-Bcl-2 antibody, anti-E-cadherin
antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9
antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody,
anti-CEA antibody, anti-retinoblastoma protein antibody, anti-ras
oncoprotein antibody, anti-Lewis X antibody, anti-Ki-67 antibody,
anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5
antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24
antibody, anti-CD10 antibody, anti-CD11c antibody, anti-CD13
antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19
antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31
antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35
antibody, anti-CD38 antibody, anti-CD41 antibody, anti-LCA/CD45
antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39
antibody, anti-CD100 antibody, anti-CD95/Fas antibody, anti-CD99
antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71
antibody, anti-c-myc antibody, anti-cytokeratins antibody,
anti-vimentins antibody, anti-HPV proteins antibody, anti-kappa
light chains antibody, anti-lambda light chains antibody,
anti-melanosomes antibody, anti-prostate specific antigen antibody,
anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin
antibody, anti-keratins antibody and anti-Tn-antigen antibody.
[0036] An "isolated" protein, such as antibody, is one which has
been identified and separated and/or recovered from a component of
its natural environment. Contaminant components of its natural
environment are materials which would interfere with diagnostic or
therapeutic uses for the protein, such as antibody, and may include
enzymes, hormones, and other proteinaceous or nonproteinaceous
solutes. In preferred embodiments, the protein, such as antibody,
will be purified (1) to greater than 95% by weight as determined by
the Lowry method, and most preferably more than 99% by weight, (2)
to a degree sufficient to obtain at least 15 residues of N-terminal
or internal amino acid sequence by use of a spinning cup
sequenator, or (3) to homogeneity by SDS-PAGE under reducing or
nonreducing conditions using Coomassie blue or, preferably, silver
stain.
[0037] The protein is preferably essentially pure and desirably
essentially homogeneous (i.e. free from contaminating proteins).
"Essentially pure" protein means a composition comprising at least
about 90% by weight of the protein, based on total weight of the
composition, preferably at least about 95% by weight.
[0038] "Essentially homogeneous" protein means a composition
comprising at least about 99% by weight of protein, based on total
weight of the composition.
[0039] The term "antibody" is used in the broadest sense and
specifically covers monoclonal antibodies (including full length
antibodies which have an immunoglobulin Fc region), antibody
compositions with polyepitopic specificity, bispecific antibodies,
diabodies, and single-chain molecules, as well as antibody
fragments (e.g., Fab, F(ab').sub.2, and Fv).
[0040] The basic 4-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains. An IgM antibody consists of 5 of the
basic heterotetramer unit along with an additional polypeptide
called a J chain, and contains 10 antigen binding sites, while IgA
antibodies comprise from 2-5 of the basic 4-chain units which can
polymerize to form polyvalent assemblages in combination with the J
chain. In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to an H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
domain (V.sub.H) followed by three constant domains (C.sub.H) for
each of the .alpha. and .gamma. chains and four C.sub.H domains for
.mu. and .epsilon. isotypes. Each L chain has at the N-terminus, a
variable domain (V.sub.L) followed by a constant domain at its
other end. The V.sub.L is aligned with the V.sub.H and the C.sub.L
is aligned with the first constant domain of the heavy chain
(C.sub.H1). Particular amino acid residues are believed to form an
interface between the light chain and heavy chain variable domains.
The pairing of a V.sub.H and V.sub.L together forms a single
antigen-binding site. For the structure and properties of the
different classes of antibodies, see e.g., Basic and Clinical
Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram
G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page
71 and Chapter 6.
[0041] The L chain from any vertebrate species can be assigned to
one of two clearly distinct types, called kappa and lambda, based
on the amino acid sequences of their constant domains. Depending on
the amino acid sequence of the constant domain of their heavy
chains (CH), immunoglobulins can be assigned to different classes
or isotypes. There are five classes of immunoglobulins: IgA, IgD,
IgE, IgG and IgM, having heavy chains designated .alpha., .delta.,
.epsilon., .gamma. and .mu., respectively. The .gamma. and .mu.
classes are further divided into subclasses on the basis of
relatively minor differences in the CH sequence and function, e.g.,
humans express the following subclasses: IgG1, IgG2, IgG3, IgG4,
IgA1 and IgA2.
[0042] The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among
antibodies. The V domain mediates antigen binding and defines the
specificity of a particular antibody for its particular antigen.
However, the variability is not evenly distributed across the
entire span of the variable domains. Instead, the V regions consist
of relatively invariant stretches called framework regions (FRs) of
about 15-30 amino acid residues separated by shorter regions of
extreme variability called "hypervariable regions" or sometimes
"complementarity determining regions" (CDRs) that are each
approximately 9-12 amino acid residues in length. The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a .beta.-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the .beta.-sheet structure. The
hypervariable regions in each chain are held together in close
proximity by the FRs and, with the hypervariable regions from the
other chain, contribute to the formation of the antigen binding
site of antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody dependent cellular cytotoxicity (ADCC). The term
"hypervariable region" (also known as "complementarity determining
regions" or CDRs) when used herein refers to the amino acid
residues of an antibody which are (usually three or four short
regions of exteme sequence variability) within the V-region domain
of an immunoglobulin which form the antigen-binding site and are
the main determinants of antigen specificity. There are at least
two methods for identifying the CDR residues: (1) An approach based
on cross-species sequence variability (i.e., Kabat et al.,
Sequences of Proteins of Immunological Interest (National Institute
of Health, Bethesda, M S 1991); and (2) An approach based on
crystallographic studies of antigen-antibody complexes (Chothia, C.
et al., J. Mol. Biol. 196: 901-917 (1987)). However, to the extent
that two residue identification techniques define regions of
overlapping, but not identical regions, they can be combined to
define a hybrid CDR.
[0043] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. In addition to their specificity, the
monoclonal antibodies are advantageous in that they are synthesized
by the hybridoma culture, uncontaminated by other immunoglobulins.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al.,
Nature, 256: 495 (1975), or may be made by recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the
techniques described in Clackson et al., Nature, 352:624-628 (1991)
and Marks et al., J. Mol. Biol., 222:581-597 (1991), for
example.
[0044] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is(are) identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, so long as they exhibit
the desired biological activity (U.S. Pat. No. 4,816,567; Morrison
et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). An
"intact" antibody is one which comprises an antigen-binding site as
well as a C.sub.L and at least the heavy chain domains, C.sub.H1,
C.sub.H2 and C.sub.H3.
[0045] An "antibody fragment" comprises a portion of an intact
antibody, preferably the antigen binding and/or the variable region
of the intact antibody. Examples of antibody fragments include Fab,
Fab', F(ab').sub.2 and Fv fragments; diabodies; linear antibodies
(see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein
Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules and
multispecific antibodies formed from antibody fragments.
[0046] Papain digestion of antibodies produced two identical
antigen-binding fragments, called "Fab" fragments, and a residual
"Fc" fragment, a designation reflecting the ability to crystallize
readily. The Fab fragment consists of an entire L chain along with
the variable region domain of the H chain (V.sub.H), and the first
constant domain of one heavy chain (C.sub.H1). Each Fab fragment is
monovalent with respect to antigen binding, i.e., it has a single
antigen-binding site. Pepsin treatment of an antibody yields a
single large F(ab').sub.2 fragment which roughly corresponds to two
disulfide linked Fab fragments having different antigen-binding
activity and is still capable of cross-linking antigen. Fab'
fragments differ from Fab fragments by having a few additional
residues at the carboxy terminus of the C.sub.H1 domain including
one or more cysteines from the antibody hinge region. Fab'-SH is
the designation herein for Fab' in which the cysteine residue(s) of
the constant domains bear a free thiol group. F(ab').sub.2 antibody
fragments originally were produced as pairs of Fab' fragments which
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0047] The Fc fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region, the
region which is also recognized by Fc receptors (FcR) found on
certain types of cells.
[0048] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This fragment
consists of a dimer of one heavy- and one light-chain variable
region domain in tight, non-covalent association. From the folding
of these two domains emanate six hypervarible loops (3 loops each
from the H and L chain) that contribute the amino acid residues for
antigen binding and confer antigen binding specificity to the
antibody. However, even a single variable domain (or half of an Fv
comprising only three CDRs specific for an antigen) has the ability
to recognize and bind antigen, although at a lower affinity than
the entire binding site.
[0049] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody fragments that comprise the VH and VL antibody domains
connected into a single polypeptide chain. Preferably, the sFv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of the sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0050] The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments (see preceding paragraph)
with short linkers (about 5-10) residues) between the V.sub.H and
V.sub.L domains such that inter-chain but not intra-chain pairing
of the V domains is achieved, thereby resulting in a bivalent
fragment, i.e., a fragment having two antigen-binding sites.
Bispecific diabodies are heterodimers of two "crossover" sFv
fragments in which the V.sub.H and V.sub.L domains of the two
antibodies are present on different polypeptide chains. Diabodies
are described in greater detail in, for example, EP 404,097; WO
93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90:
6444-6448 (1993).
[0051] An antibody "which binds" a molecular target or an antigen
of interest is one capable of binding that antigen with sufficient
affinity such that the antibody is useful in targeting a cell
expressing the antigen.
[0052] An antibody that "specifically binds to" or is "specific
for" a particular polypeptide or an epitope on a particular
polypeptide is one that binds to that particular polypeptide or
epitope on a particular polypeptide without substantially binding
to any other polypeptide or polypeptide epitope. In such
embodiments, the extent of binding of the antibody to these other
polypeptides or polypeptide epitopes will be less than 10%, as
determined by fluorescence activated cell sorting (FACS) analysis
or radioimmunoprecipitation (RIA), relative to binding to the
target polypeptide or epitope.
[0053] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) of mostly human
sequences, which contain minimal sequence derived from non-human
immunoglobulin. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
hypervariable region (also CDR) of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
"humanized antibodies" as used herein may also comprise residues
which are found neither in the recipient antibody nor the donor
antibody. These modifications are made to further refine and
optimize antibody performance. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. For further
details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et
al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992).
[0054] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: C1q binding and complement dependent
cytotoxicity; Fc receptor binding; antibody--dependent
cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of
cell surface receptors (e.g., B cell receptors); and B cell
activation.
[0055] "Antibody-dependent cell-mediated cytotoxicity" or ADCC
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g., natural
killer (NK) cells, neutrophils and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are required for
killing of the target cell by this mechanism. The primary cells for
mediating ADCC, NK cells, express Fc.gamma.RIII only, whereas
monocytes express Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. Fc
expression on hematopoietic cells is summarized in Table 3 on page
464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991). To
assess ADCC activity of a molecule of interest, an in vitro ACDD
assay, such as that described in U.S. Pat. No. 5,500,362 or
5,821,337 may be performed. Useful effector cells for such assays
include peripheral blood mononuclear cells (PBMC) and natural
killer (NK) cells. Alternatively, or additionally, ADCC activity of
the molecule of interest may be assessed in vivo, e.g., in an
animal model such as that disclosed in Clynes et al., PNAS USA
95:652-656 (1998).
[0056] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native
sequence human FcR. Moreover, a preferred FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors, Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see M. Daeron, Annu. Rev.
Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet,
Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods
4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41
(1995). Other FcRs, including those to be identified in the future,
are encompassed by the term "FcR" herein. The term also includes
the neonatal receptor, FcRn, which is responsible for the transfer
of maternal IgGs to the fetus. Guyer et al., J. Immunol. 117: 587
(1976) and Kim et al., J. Immunol. 24: 249 (1994).
[0057] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils, with PBMCs and MNK
cells being preferred. The effector cells may be isolated from a
native source, e.g., blood.
[0058] "Complement dependent cytotoxicity" of "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), may be
performed.
[0059] The terms "conjugate," "conjugated," and "conjugation" refer
to any and all forms of covalent or non-covalent linkage, and
include, without limitation, direct genetic or chemical fusion,
coupling through a linker or a cross-linking agent, and
non-covalent association, for example using a leucine zipper.
Antibody conjugates have another entity, such as a cytotoxic
compound, drug, composition, compound, radioactive element, or
detectable label, attached to an antibody or antibody fragment.
[0060] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented.
[0061] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, non-human higher
primates, domestic and farm animals, and zoo, sports, or pet
animals, such as dogs, horses, rabbits, cattle, pigs, hamsters,
mice, cats, etc. Preferably, the mammal is human.
[0062] A "disorder" is any condition that would benefit from
treatment with the protein. This includes chronic and acute
disorders or diseases including those pathological conditions which
predispose the mammal to the disorder in question.
[0063] A "therapeutically effective amount" is at least the minimum
concentration required to effect a measurable improvement or
prevention of a particular disorder.
[0064] Therapeutically effective amounts of known proteins are well
known in the art, while the effective amounts of proteins
hereinafter discovered may be determined by standard techniques
which are well within the skill of a skilled artisan, such as an
ordinary physician.
II. Modes for Carrying Out the Invention
[0065] A. Protein Preparation
[0066] In accordance with the present invention, the protein is
produced by recombinant DNA techniques, i.e., by culturing cells
transformed or transfected with a vector containing nucleic acid
encoding the protein, as is well known in art.
[0067] Preparation of the protein by recombinant means may be
accomplished by transfecting or transforming suitable host cells
with expression or cloning vectors and cultured in conventional
nutrient media modified as appropriate for inducing promoters,
selecting transformants, or amplifying the genes encoding the
desired sequences. The culture conditions, such as media,
temperature, pH and the like, can be selected by the skilled
artisan without undue experimentation. In general, principles,
protocols, and practical techniques for maximizing the productivity
of cell cultures can be found in Mammalian Cell Biotechnology: A
Practical Approach, M. Butler, Ed. (IRL Press, 1991) and Sambrook
et al., Molecular Cloning: A Laboratory Manual, New York: Cold
Spring Harbor Press. Methods of transfection are known to the
ordinarily skilled artisan, and include for example, CaPO.sub.4 and
CaCl.sub.2 transfection, electroporation, microinjection, etc.
Suitable techniques are also described in Sambrook et al., supra.
Additional transfection techniques are described in Shaw et al.,
Gene 23: 315 (1983); WO 89/05859; Graham et al., Virology 52:
456-457 (1978) and U.S. Pat. No. 4,399,216.
[0068] The nucleic acid encoding the desired protein may be
inserted into a replicable vector for cloning or expression.
Suitable vectors are publicly available and may take the form of a
plasmid, cosmid, viral particle or phage. The appropriate nucleic
acid sequence may be inserted into the vector by a variety of
procedures. In general, DNA is inserted into an appropriate
restriction endonuclease site(s) using techniques known in the art.
Vector components generally include, but are not limited to, one or
more of a signal sequence, an origin of replication, one or more
marker genes, and enhancer element, a promoter, and a transcription
termination sequence. Construction of suitable vectors containing
one or more of these components employs standard ligation
techniques which are known to the skilled artisan.
[0069] Forms of the protein may be recovered from culture medium or
from host cell lysates. If membrane-bound, it can be released from
the membrane using a suitable detergent or through enzymatic
cleavage. Cells employed for expression can also be disrupted by
various physical or chemical means, such as freeze-thaw cycling,
sonication, mechanical disruption or cell lysing agents.
[0070] Purification of the protein may be effected by any suitable
technique known in the art, such as for example, fractionation on
an ion-exchange column, ethanol precipitation, reverse phase HPLC,
chromatography on silica or cation-exchange resin (e.g., DEAE),
chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel
filtration using protein A Sepharose columns (e.g., SEPHADEX.RTM.
G-75) to remove contaminants such as IgG, and metal chelating
columns to bind epitope-tagged forms.
[0071] B. Antibody Preparation
[0072] In certain embodiments of the invention, the protein of
choice is an antibody. Techniques for the production of antibodies,
including polyclonal, monoclonal, humanized, bispecific and
heteroconjugate antibodies follow. [0073] (i) Polyclonal
Antibodies.
[0074] Polyclonal antibodies are generally raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor.
Examples of adjuvants which may be employed include Freund's
complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,
synthetic trehalose dicorynomycolate). The immunization protocol
may be selected by one skilled in the art without undue
experimentation.
[0075] One month later the animals are boosted with 1/5 to 1/10 the
original amount of peptide or conjugate in Freund's complete
adjuvant by subcutaneous injection at multiple sites. Seven to 14
days later the animals are bled and the serum is assayed for
antibody titer. Animals are boosted until the titer plateaus.
Preferably, the animal is boosted with the conjugate of the same
antigen, but conjugated to a different protein and/or through a
different cross-linking reagent. Conjugates also can be made in
recombinant cell culture as protein fusions. Also, aggregating
agents such as alum are suitably used to enhance the immune
response. [0076] (ii) Monoclonal Antibodies.
[0077] Monoclonal antibodies are obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies.
[0078] For example, the monoclonal antibodies may be made using the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (U.S. Pat. No.
4,816,567).
[0079] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as hereinabove described to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes then are fused with myeloma cells using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986).
[0080] The immunizing agent will typically include the protein to
be formulated. Generally either peripheral blood lymphocytes
("PBLs") are used if cells of human origin are desired, or spleen
cells or lymph node cells are used if non-human mammalian sources
are desired. The lymphoctyes are then fused with an immortalized
cell line using a suitable fusing agent, such as polyethylene
glycol, to form a hybridoma cell. Goding, Monoclonal antibodies:
Principles and Practice, Academic Press (1986), pp. 59-103.
Immortalized cell lines are usually transformed mammalian cell,
particularly myeloma cells of rodent, bovine and human origin.
Usually, rat or mouse myeloma cell lines are employed. The
hybridoma cells thus prepared are seeded and grown in a suitable
culture medium that preferably contains one or more substances that
inhibit the growth or survival of the unfused, parental myeloma
cells. For example, if the parental myeloma cells lack the enzyme
hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT),
the culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium), which
substances prevent the growth of HGPRT-deficient cells.
[0081] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 cells available from the American
Type Culture Collection, Rockville, Md. USA. Human myeloma and
mouse-human heteromyeloma cell lines also have been described for
the production of human monoclonal antibodies (Kozbor, J. Immunol.,
133:3001 (1984); Brodeur et al., Monoclonal Antibody Production
Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New
York, 1987)).
[0082] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA).
[0083] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis of Munson et al.,
Anal. Biochem., 107:220 (1980).
[0084] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, supra). Suitable culture media for this
purpose include, for example, D-MEM or RPMI-1640 medium. In
addition, the hybridoma cells may be grown in vivo as ascites
tumors in an animal.
[0085] The immunizing agent will typically include the epitope
protein to which the antibody binds. Generally, either peripheral
blood lymphocytes ("PBLs") are used if cells of human origin are
desired, or spleen cells or lymph node cells are used if non-human
mammalian sources are desired. The lymphocytes are then fused with
an immortalized cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell. Goding, Monoclonal
Antibodies: Principals and Practice, Academic Press (1986), pp.
59-103.
[0086] Immortalized cell lines are usually transformed mammalian
cells, particularly myeloma cells of rodent, bovine and human
origin. Usually, rat or mouse myelome cell lines are employed. The
hybridoma cells may be cultured in a suitable culture medium that
preferably contains one or more substances that inhibit the growth
or survival of the unfused, immortalized cells. For example, if the
parental cells lack the enzyme hypoxanthine guanine phosphoribosyl
transferase (HGPRT or HPRT), the culture medium for the hybridomas
typically will include hypoxanthine, aminopterin and thymidine
("HAT medium"), which substances prevent the growth of
HGPRT-deficient cells.
[0087] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Rockville, Md. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies. Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63.
[0088] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the protein to be formulated. Preferably, the
binding specificity of monoclonal antibodies produced by the
hybridoma cells is determined by immunoprecipitation or by an in
vitro binding assay, such as radioimmunoassay (RIA) or
enzyme-linked immunoabsorbent assay (ELISA). Such techniques and
assays are known in the art. The binding affinity of the monoclonal
antibody can, for example, be determined by the Scatchard analysis
of Munson and Pollard, Anal. Biochem., 107:220 (1980).
[0089] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods. Goding, supra. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0090] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0091] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do
not otherwise produce immunoglobulin protein, to obtain the
synthesis of monoclonal antibodies in the recombinant host cells.
Review articles on recombinant expression in bacteria of DNA
encoding the antibody include Skerra et al., Curr. Opinion in
Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs.
130:151-188 (1992).
[0092] In a further embodiment, antibodies can be isolated from
antibody phage libraries generated using the techniques described
in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al.,
Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,
222:581-597 (1991) describe the isolation of murine and human
antibodies, respectively, using phage libraries. Subsequent
publications describe the production of high affinity (nM range)
human antibodies by chain shuffling (Marks et al., Bio/Technology,
10:779-783 (1992)), as well as combinatorial infection and in vivo
recombination as a strategy for constructing very large phage
libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266
(1993)). Thus, these techniques are viable alternatives to
traditional monoclonal antibody hybridoma techniques for isolation
of monoclonal antibodies.
[0093] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy- and light-chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851
(1984)), or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide.
[0094] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody, or they are
substituted for the variable domains of one antigen-combining site
of an antibody to create a chimeric bivalent antibody comprising
one antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0095] Chimeric or hybrid antibodies also may be prepared in vitro
using known methods in synthetic protein chemistry, including those
involving crosslinking agents. For example, immunotoxins may be
constructed using a disulfide-exchange reaction or by forming a
thioether bond. Examples of suitable reagents for this purpose
include iminothiolate and methyl-4-mercaptobutyrimidate. [0096]
(iii) Humanized and Human Antibodies.
[0097] The antibodies subject to the formulation method may further
comprise humanized or human antibodies. Humanized forms of
non-human (e.g., murine) antibodies are chimeric immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab',
F(ab').sub.2 or other antigen-binding subsequences of antibodies)
which contain minimal sequence derived from non-human
immunoglobulin. Humanized antibodies include human immunoglobulins
(recipient antibody) in which residues from a complementarity
determining region (CDR) of the recipient are replaced by residues
from a CDR of a non-human species (donor antibody) such as mouse,
rat or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Humanized antibodies may also comprise residues which are found
neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domain, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. Jones et
al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332:
323-329 (1988) and Presta, Curr. Opin. Struct. Biol. 2: 593-596
(1992).
[0098] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers, Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);
Verhoeyen et al., Science 239:1534-1536 (1988), or through
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0099] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework (FR) for the
humanized antibody. Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987). Another method uses
a particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies. Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285
(1992); Presta et al., J. Immnol., 151:2623 (1993).
[0100] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
CDR residues are directly and most substantially involved in
influencing antigen binding.
[0101] Alternatively, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno.,
7:33 (1993). Human antibodies can also be derived from
phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381
(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)).
[0102] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries.
Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991); Marks et al.,
J. Mol. Biol. 222: 581 (1991). The techniques of Cole et al., and
Boerner et al., are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol. 147(1): 86-95 (1991). Similarly, human antibodies can be
made by introducing human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resemble that
seen in human in all respects, including gene rearrangement,
assembly and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806, 5,569,825,
5,625,126, 5,633,425, 5,661,016 and in the following scientific
publications: Marks et al., Bio/Technology 10: 779-783 (1992);
Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368:
812-13 (1994), Fishwild et al., Nature Biotechnology 14: 845-51
(1996), Neuberger, Nature Biotechnology 14: 826 (1996) and Lonberg
and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995). [0103] (iv)
Antibody Dependent Enzyme-Mediated Prodrug Therapy (ADEPT)
[0104] The antibodies of the present invention may also be used in
ADEPT by conjugating the antibody to a prodrug-activating enzyme
which converts a prodrug (e.g. a peptidyl chemotherapeutic agent,
see WO 81/01145) to an active anti-cancer drug. See, for example,
WO 88/07378 and U.S. Pat. No. 4,975,278.
[0105] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such as way
so as to convert it into its more active, cytotoxic form.
[0106] Enzymes that are useful in the method of this invention
include, but are not limited to, glycosidase, glucose oxidase,
human lysozyme, human glucuronidase, alkaline phosphatase useful
for converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful for converting non-toxic
5-fluorocytosine into the anti-cancer drug 5-fluorouracil;
proteases, such as serratia protease, thermolysin, subtilisin,
carboxypeptidases (e.g., carboxypeptidase G2 and carboxypeptidase
A) and cathepsins (such as cathepsins B and L), that are useful for
converting peptide-containing prodrugs into free drugs;
D-alanylcarboxypeptidases, useful for converting prodrugs that
contain D-amino acid substituents; carbohydrate-cleaving enzymes
such as .beta.-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; .beta.-lactamase
useful for converting drugs derivatized with .beta.-lactams into
free drugs; and penicillin amidases, such as penicillin Vamidase or
penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes" can be used
to convert the prodrugs of the invention into free active drugs
(see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzyme
conjugates can be prepared as described herein for delivery of the
abzyme to a tumor cell population.
[0107] The enzymes of this invention can be covalently bound to the
anti-IL-17 or anti-LIF antibodies by techniques well known in the
art such as the use of the heterobifunctional cross-linking agents
discussed above. Alternatively, fusion proteins comprising at least
the antigen binding region of the antibody of the invention linked
to at least a functionally active portion of an enzyme of the
invention can be constructed using recombinant DNA techniques well
known in the art (see, e.g. Neuberger et al., Nature 312: 604-608
(1984)). [0108] (iv) Bispecific and Polyspecific Antibodies
[0109] Bispecific antibodies (BsAbs) are antibodies that have
binding specificities for at least two different epitopes. Such
antibodies can be derived from full length antibodies or antibody
fragments (e.g. F(ab').sub.2bispecific antibodies).
[0110] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities.
Millstein et al., Nature, 305:537-539 (1983). Because of the random
assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829 and in Traunecker et al., EMBO J., 10:3655-3659
(1991).
[0111] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, C.sub.H2, and C.sub.H3 regions. It is
preferred to have the first heavy-chain constant region (C.sub.H1)
containing the site necessary for light-chain binding present in at
least one of the fusions. DNAs encoding the immunoglobulin
heavy-chain fusions, and, if desired, the immunoglobulin light
chain, are inserted into separate expression vectors, and are
co-transfected into a suitable host organism. For further details
of generating bispecific antibodies, see, for example, Suresh et
al., Methods in Enzymology 121: 210 (1986).
[0112] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3
regions. It is preferred to have the first heavy-chain constant
region (CH1) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0113] According to another approach described in WO 96/27011, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers which are recovered from
recombinant cell culture. The preferred interface comprises at
least a part of the CH3 region of an antibody constant domain. In
this method, one or more small amino acid side chains from the
interface of the first antibody molecule are replaced with larger
side chains (e.g., tyrosine or tryptophan). Compensatory "cavities"
of identical or similar size to the large side chains(s) are
created on the interface of the second antibody molecule by
replacing large amino acid side chains with smaller ones (e.g.,
alanine or threonine). This provides a mechanism for increasing the
yield of the heterodimer over other unwanted end-products such as
homodimers.
[0114] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690, published Mar. 3, 1994. For
further details of generating bispecific antibodies see, for
example, Suresh et al., Methods in Enzymology, 121:210 (1986).
[0115] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373).
Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0116] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. The
following techniques can also be used for the production of
bivalent antibody fragments which are not necessarily bispecific.
For example, Fab' fragments recovered from E. coli can be
chemically coupled in vitro to form bivalent antibodies. See,
Shalaby et al., J. Exp. Med., 175:217-225 (1992).
[0117] Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab').sub.2 bispecific
antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab').sub.2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-TNB derivative to form
the bispecific antibody. The bispecific antibodies produced can be
used as agents for the selective immobilization of enzymes.
[0118] Fab' fragments may be directly recovered from E. coli and
chemically coupled to form bispecific antibodies. Shalaby et al.,
J. Exp. Med. 175: 217-225 (1992) describes the production of fully
humanized bispecific antibody F(ab').sub.2 molecules. Each Fab'
fragment was separately secreted from E. coli and subjected to
directed chemical coupling in vitro to form the bispecific
antibody. The bispecific antibody thus formed was able to bind to
cells overexpressing the ErbB2 receptor and normal human T cells,
as well as trigger the lytic activity of human cytotoxic
lymphocytes against human breast tumor targets.
[0119] Various techniques for making and isolating bivalent
antibody fragments directly from recombinant cell culture have also
been described. For example, bivalent heterodimers have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. The "diabody" technology described by
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)
has provided an alternative mechanism for making
bispecific/bivalent antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific/bivalent antibody fragments by the use of
single-chain Fv (sFv) dimers has also been reported. See Gruber et
al., J. Immunol., 152:5368 (1994).
[0120] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147: 60 (1991).
[0121] Exemplary bispecific antibodies may bind to two different
epitopes on a given molecule. Alternatively, an anti-protein arm
may be combined with an arm which binds to a triggering molecule on
a leukocyte such as a T-cell receptor molecule (e.g., CD2, CD3,
CD28 or B7), or Fc receptors for IgG (Fc.gamma.R), such as
Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16) so
as to focus cellular defense mechanisms to the cell expressing the
particular protein. Bispecific antibocis may also be used to
localize cytotoxic agents to cells which express a particular
protein. Such antibodies possess a protein-binding arm and an arm
which binds a cytotoxic agent or a radionuclide chelator, such as
EOTUBE, DPTA, DOTA or TETA. Another bispecific antibody of interest
binds the protein of interest and further binds tissue factor (TF).
[0122] (v) Heteroconjugate Antibodies
[0123] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to tatget immune system cells to unwanted cells, U.S.
Pat. No. 4,676,980, and for treatment of HIV infection. WO
91/00360, WO 92/200373 and EP 03089. It is contemplated that the
antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0124] C. Purification of the Proteins, Including Antibodies
[0125] When the target polypeptide is expressed in a recombinant
cell other than one of human origin, the target polypeptide is
completely free of proteins or polypeptides of human origin.
However, it is necessary to purify the target polypeptide from
recombinant cell proteins or polypeptides to obtain preparations
that are substantially homogeneous as to the target polypeptide. As
a first step, the culture medium or lysate is typically centrifuged
to remove particulate cell debris. The membrane and soluble protein
fractions are then separated. The target polypeptide may then be
purified from the soluble protein fraction and from the membrane
fraction of the culture lysate, depending on whether the target
polypeptide is membrane bound. The following procedures are
exemplary of suitable purification procedures: fractionation on
immunoaffinity or ion-exchange columns; ethanol precipitation;
reverse phase HPLC; chromatography on silica or on a cation
exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium
sulfate precipitation; gel filtration using, for example, Sephadex
G-75; and protein A Sepharose columns to remove contaminants such
as IgG.
[0126] Most companies currently producing monoclonal antibodies
(MAbs) use a three-column platform approach comprising Protein A
affinity chromatography for product capture, followed by anion
exchange chromatography in flow-through mode to extract negatively
charged contaminants such as host cell protein (HCP), endotoxins,
host DNA, and leached Protein A, and then cation exchange
chromatography or hydrophobic interaction chromatography (HIC) in
retention mode to remove positively charged contaminant species
including residual HCP and product aggregates.
[0127] Those viruses that may be present in protein solutions are
larger than the proteins themselves. It is thus presumed that
viruses can be removed from proteins in accordance with size, by
filtration.
[0128] Virus filtration can remove larger, e.g., retroviruses
(80-100 nm diameter), typically using high throughput membranes
with nominal pore size of about 60 nm. Since high throughput
membranes with nominal pore size of 20 nm are also commercially
available, it is possible to remove smaller viruses by filtration,
such as, for example, parvoviruses (18-26 nm diameter), while
allowing passage of proteins that are as large as 160 kD (.about.8
nm), e.g., monoclonal antibodies. The present invention is
primarily intended for resolving issues typically associated with
the filtration of such smaller viruses, using viral removal filters
of smaller pore size.
[0129] Typically, a virus filtration step can be implemented at any
one of several points in a given downstream process. For example,
in a typical monoclonal antibody purification process, virus
filtration may take place following a low pH viral inactivation
step, or following an intermediate column chromatography step, or
after a final column chromatography step.
[0130] According to the invention, virus filtration unit operation
could be carried out at any stage in the downstream process. Virus
filtration during downstream processing of monoclonal antibody is
typically performed after an affinity chromatography step (capture
step) and an ion-exchange purification step (polishing step).
[0131] The experimental setup used in the experiments disclosed
herein is illustrated in FIG. 1. It is emphasized, however, that
the invention is not so limited. Other arrangements, well known in
the art, are also suitable and can be used in the methods of the
present invention.
[0132] In tangential flow virus filtration, the protein solution is
usually pumped around at a constant rate of flow on the retention
side. The differential pressure generated across the virus removal
filter, allows protein solution to permeate through the filter
while the viruses are retained on the retentate side.
[0133] In the case of so called "normal-flow" or "dead-end" virus
filtration, the same virus filter as that used in tangential virus
filtration can be used, although the peripheral equipment and
operating procedures are much simpler and less expensive than in
the case of tangential flow virus filtration. Thus, in principle,
"normal-flow" filtration involves placing the
macromolecule-containing solution in a pressure vessel prior to
filtration and pressing the solution through the virus removal
filter with the aid of a pressure source, suitably nitrogen (gas)
or air. Alternatively, a pump could be used on the retentate side
to filter the liquid through the virus removal filter at a
pre-determined flow rate.
[0134] The degree of fineness of filters generally, is normally
expressed as pore size or the approximate molecular weight
(relative molecular mass) at which the molecules are stopped by the
filter, the so called cut-off.
[0135] Virus filters are known in the art and are supplied by
Millipore from Massachusetts, USA and Asahi Chemical Industry Co.,
Ltd. from Japan, among others. Suitable parvovirus retentive
filters include VIRESOLVE.RTM. Pro (Millipore Corp., Billerica,
Mass.) VIRESOLVE.RTM. Pro membrane has an asymmetric dual layer
structure and is made from polyethersulfone (PES). The membrane
structure is designed to retain viruses greater than 20 nm in size
while allowing proteins of molecular weight less than 180 kDa to
permeate through the membrane. Other filters suitable for the
removal of small viruses, including parvoviruses, from protein
solutions include NOVASIP.TM. DV20 and DV50 Virus Removal Filter
Capsules (Pall Corp., East Hills, N.Y.), VIROSART.RTM. CPV, Planova
20 N (Asahi Kasei) and BioEX (Asahi Kasei). The NOVASIP.TM. DV20
grade capsule filter utilizes an ULTIPOR.RTM. VF-grade DV20 grade
pleated membrane cartridge to remove parvoviruses and other viruses
as small as 20 nm from protein solutions up to 5-10 liters. The
NOVASIP.TM. DV50 grade capsule filter incorporates an ULTIPOR.RTM.
VF DV50 grade ULTIPLEAT.RTM. membrane cartridge for removal of
viruses 40-50 nm and larger. NOVASIP.RTM. ULTIPOR.RTM. VF capsule
filters are supplied non-sterile and can also be Gamma-irradiated.
VIROSART.RTM. CPV utilizes double--layer polyethersulfone
asymmetric membrane and retains more than 4 log of parvoviruses and
6 log of retroviruses.
[0136] Prefiltration of the feed solution can have a dramatic
impact on filter performance Prefiltration typically is targeted to
remove impurities and contaminants that might lead to fouling of
virus filters, such as protein aggregates, DNA and other trace
materials.
[0137] According to the present invention, a striking enhancement
of the efficacy of virus filters can be achieved by a prefiltration
step including the use of both cation exchange and endotoxin
removal media. In this context, the term "medium" or "media" is
used to cover any means for performing the cation exchange and
endotoxin removal steps, respectively. Thus, the term "cation
exchange medium" specifically includes, without limitation, cation
exchange resins, matrices, absorbers, and the like. The term
"endotoxin removal medium" includes, without limitation, any
positively charged membrane surface, including, for example,
chromatographic endotoxin removal media, endotoxin affinity removal
media, and the like.
[0138] Cation exchange media suitable for use in the prefiltration
step of the present invention include, without limitation,
MUSTANG.RTM. S, SARTOBIND.RTM. S, VIRESOLVE.RTM. Shield, SPFF,
SPXL, CAPTO.RTM. S, POROS.RTM. 50 HS, FRACTOGEL.RTM. S,
HYPERCEL.RTM. D etc., which are commercially available.
[0139] Endotoxin removal media suitable for use in the
prefiltration step of the present invention include, without
limitation, MUSTANG.RTM. E, MUSTANG.RTM. Q, SARTOBIND.RTM. Q,
CHROMASORB.RTM., POSSIDYNE.RTM., CAPTO.RTM. Q, QSFF, POROS.RTM. Q,
FRACTOGEL.RTM. Q etc., which are commercially available.
[0140] The pre-filtration step can be performed, for example, by
taking the in process chromatography pool and processing the pool
over a filtration train that comprises the endotoxin removal and
cation exchange media and parvovirus filter. The endotoxin removal
and cation exchange media act as pre-filtration steps and the
capacity of parvovirus filter is independent of the sequence of two
steps in the filtration train. The filtration train can work
continuously as a single step or it can be operated as different
unit operations. For example, in one embodiment, the chromatography
pool is first processed over endotoxin removal media, the collected
pool is then processed over cation exchange media and the
subsequent pool is filtered with parvovirus filter. As mentioned
above, the order of applying the cation exchange media and
endotoxin removal media in the process sequence does not impact
parvovirus filtration capacity. The process can be operated over a
wide pH range, such as, for example, in the pH range of 4-10, with
optimal filter capacity being dependent on the target impurity
profile and product attributes. Similarly, protein concentrations
can vary over a wide range, such as, for example, 1-40 g/L, and
does not limit the mass throughput of parvovirus filters.
[0141] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention. All citations throughout the
disclosure are hereby expressly incorporated by reference.
EXAMPLE
[0142] Materials and Methods [0143] 1. Protein Solution
[0144] Since virus filtration during downstream processing of
monoclonal antibody is performed after the affinity chromatography
(capture step) and an ion-exchange step (polishing step), all
filtration experiments were performed with commercially relevant in
process ion exchange (cation or anion-exchange) chromatography
pools. The mAb concentration and pool conductivity for cation
exchange and anion exchange pools were respectively 10 mg/ml and 10
mS/cm and 8 mg/ml and 4 ms/cm. Filtration experiments were
performed either with fresh feedstock (used within 24 hours of
production) or with feedstock that was frozen at -70.degree. C.
after production and was thawed at 4-8.degree. C. prior to use. No
significant difference was seen in results obtained with fresh or
freeze-thawed feedstock. Protein concentration was determined using
a UV-vis spectrophotometer (NanoDrop ND-1000, NanoDrop
Technologies, Wilmington, Del.) with absorbance measured at 280 nm.
[0145] 2. Membranes
[0146] Filtration experiments were performed with VIRESOLVE.RTM.
Pro (Millipore Corp., Billerica, Mass.) parvovirus retentive
filter. VIRESOLVE.RTM. Pro membrane has an asymmetric dual layer
structure and is made from polyethersulfone (PES). The membrane
structure is designed to retain viruses greater than 20 nm in size
while allowing proteins of molecular weight less than 180 kDa to
permeate through the membrane. Prefilters to VIRESOLVE.RTM. Pro
evaluated in this study included VIRESOLVE.RTM. Optiscale 40 depth
filter (Millipore Corp., Billerica, Mass.), FLUORODYNE.RTM. EX Mini
0.2 .mu.m sterile filter (Pall Corp., East Hills, N.Y.) and the
membrane adsorbers from MUSTANG.RTM. family (Pall Corp., East
Hills, N.Y.). The membrane adsorbers were procured from the vendor
in fully encapsulated ACRODISC.RTM. units. Table 1 summarizes the
key properties (functional group, bed volume, pore size etc.) of
all the pre-filters used in this study.
TABLE-US-00001 TABLE 1 Key Properties of Prefilters Bed Volume/
Pore Prefilter Utility Base Matrix Functional Group Surface Area
Size VIRESOLVE .RTM. Depth Filter Diatomaceous Earth -- -- --
FLURODYNE .RTM. EX Sterile Filter Polyether sulfone -- 3.8 cm2 0.2
.mu.m MUSTANG .RTM. S Strong Cation Exchanger Polyether sulfone
Sulfonic Acid 0.18 ml 0.8 .mu.m MUSTANG .RTM. Q Strong Anion
Exchanger Polyether sulfone Quaternary Amine 0.18 ml 0.8 .mu.m
MUSTANG .RTM. E Endotoxin Removal Polyether sulfone Polyethylene
Imine 0.12 ml 0.2 .mu.m
[0147] 3. Experimental Setup
[0148] Filtration experiments were performed with a custom-built
apparatus shown in FIG. 1. The load material, i.e., in process mAb
pool, was placed in the load reservoir and was filtered through a
filtration train consisting of different combinations of
pre-filters and commercially available parvovirus filters. In all
filtration experiments, the constant filtration flow rate
(P.sub.max) method was used. Pressure transducers were placed
upstream of each filter and were coupled to a Millidaq or a Netdaq
system to record differential pressure data as a function of time
or mass throughput. Filtrate from the parvovirus filter was
collected in a reservoir, which was kept on a load cell to record
mass throughput as a function of time.
[0149] Results and Discussion
[0150] Downstream purification of mAbs expressed in mammalian cell
cultures typically utilize centrifugation and depth filtration as a
first step to remove cells and cell debris, followed by affinity
chromatography for mAb capture and removal of host cell proteins
(HCP), followed by cation exchange chromatography, virus
filtration, and anion exchange chromatography for further removal
of impurities such as aggregates, viruses, leached protein A and
HCP's. Majority of the experiments in this study were performed
with cation exchange pool with cation exchange chromatography being
the second chromatography step.
[0151] FIG. 2 shows the experimental data for differential pressure
across Viresolve Pro at a constant flux of 200 L/m.sup.2/hr with a
therapeutic mAb feed stream with different prefilters. X-axis
represents the mass of mAb loaded per square meter of virus filter.
Y-axis represents the differential pressure across the virus filter
as a function of mass throughput. The data clearly indicates that
the depth filter provides several orders of magnitude increase in
virus filtration capacity compared to sterile filter. Similar
observations were made by Bolton et al. (Bolton et al. Appl.
Biochem. 43:55-63, 2006) when evaluating the effect of VIRESOLVE
Prefilter.TM.--a depth filter media--as a pre-filter to NFP
parvovirus retentive filter (Millipore Corp.) with a polyclonal IgG
solution. The authors attributed the increase in capacity to the
selective adsorption of foulant--denatured protein--due to
hydrophobic interactions.
[0152] Although depth filters have traditionally been used
successfully for clarification of cell culture fluid, there are
quite a few limitations that deserve extra consideration when used
downstream of capture steps, e.g., as a prefilter to parvovirus
retentive filter. [0153] (a) Depth filters are not base stable
which prevents the sanitization of process train after
installation, resulting in open processing and potential for
bioburden growth. [0154] (b) Composition of depth filters includes
diatomaceous earth as a key component, which is typically food
grade and presents quality concerns. [0155] (c) The diatomaceous
earth is generally sourced from nature--lacking a well defined
chemical process--and can thus can have lot to lot variations.
[0156] (d) Depth filters also tend to leach metals, beta-glycans
and other impurities, the clearance of which needs to be
demonstrated and validated with downstream operations.
[0157] These limitations put extra burden on process development as
the unit operations downstream of depth filter would have to be
designed to provide adequate clearance of leachables. However, even
if the requisite of leachable clearance was met, there are reasons
to be concerned that a particular lot of depth filter may have
significantly higher leachables than what the process is capable of
clearing as the key components are sourced from nature, that is,
they lack a well defined chemical synthesis process.
[0158] There has thus been a significant interest in development of
pre-filters that do not present these limitations. As mentioned
above, Brown et al. (Brown et al. IBC's 20th Antibody Development
and Production, San Diego, Calif., 2008) recently showed that
Mustang S, a strong negatively charged ion-exchanger, when used as
a prefilter could increase the capacity of parvovirus retentive
filter by several fold. Experiments were thus conducted to evaluate
the effect of different prefiltration media to Viresolve.RTM. Pro.
The experimental data at pH 5.0 and 6.5 is shown in FIGS. 3 (a)
& (b). The data shows that while cation exchange media shows
slight benefit over endotoxin removal adsorber at pH 5.0, the
benefit disappeared at pH 6.5. While the overall capacities with
both media were higher than those with sterile filter (FIG. 2);
they were nonetheless significantly short of the capacity required
to successfully conduct the unit operation at manufacturing
scale.
[0159] Based on the hypothesis that both cation exchange and
endotoxin removal media could be removing two different foulants;
both of which may lead to filter fouling, an experiment was
designed with a novel prefiltration train that included both cation
exchange and endotoxin removal media. Experimental results are
shown in FIGS. 4 (a) and (b) at pH 5.0 and pH 6.5 respectively. The
data clearly indicate that the combination of two media is
significantly better than each of the media by itself. For example,
at pH 5.0, the combination of cation exchange and endotoxin removal
media provide greater than an order of magnitude improvement in
capacity at 20 psi differential pressure. While similar trend was
also seen at pH 6.5, the overall capacity was lower than that
obtained at pH 5.0. It could be due to more robust removal of
impurities at lower pH.
[0160] Experimental results with MAb2 are shown in FIG. 5.
Consistent with data in FIG. 4, the novel prefiltration train
containing both endotoxin removal media and cation exchange media
increased the capacity substantially, suggesting that endotoxin
removal media and cation exchange media work synergistically and
remove two different classes of foulants.
CONCLUSIONS
[0161] Majority of the previous work has focused on the use of
depth filters or cation exchange membrane adsorbers as prefilters
to increase the capacity of parvovirus retentive filters. While
depth filters provide a robust mechanism for increasing virus
filtration capacity, limitations associated with them such as
leachables limit their application to a specific stage in the
downstream purification sequence. While cation-exchange membrane
adsorbers may increase the parvovirus filter capacity for some
monoclonal antibody (mAb) feedstreams, they may not be universally
applicable as seen with the data in this study, suggesting that
there may be multiple foulants present, which need to be addressed
to further improve performance of parvovirus removal filters.
[0162] The present invention, as demonstrated by the above
experimental results, highlights two aspects--(1) Endotoxin removal
media by itself can effectively increase the capacity of parvovirus
filters when used for prefiltration and (2) coupling of endotoxin
removal and cation exchange media in the prefiltration train can
provide several-fold increase in parvovirus filtration capacity,
lowering raw material costs and facilitating successful operation
of virus filtration at manufacturing scale.
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