U.S. patent application number 12/491677 was filed with the patent office on 2010-04-08 for use of flow-cytrometric analysis to optimize cell banking strategies for cho cells.
This patent application is currently assigned to Boehringer Ingelheim Pharma GmbH & Co. KG. Invention is credited to Juergen FIEDER, Hitto KAUFMANN, Ralf OTTO.
Application Number | 20100086947 12/491677 |
Document ID | / |
Family ID | 36118077 |
Filed Date | 2010-04-08 |
United States Patent
Application |
20100086947 |
Kind Code |
A1 |
KAUFMANN; Hitto ; et
al. |
April 8, 2010 |
USE OF FLOW-CYTROMETRIC ANALYSIS TO OPTIMIZE CELL BANKING
STRATEGIES FOR CHO CELLS
Abstract
Production of biopharmaceuticals from CHO cells requires
generation of master-, working- and post-production cell banks of
high quality, partly under GMP conditions. An optimal
cryopreservation strategy is needed for each new production cell
line, particularly with regard to the desire to establish
production processes that are completely devoid of serum or even
any animal components and to ensure robust thaw performance for
reliable production. Here we describe a novel strategy employing
flow cytometric (FC) analysis of Annexin V-stained cells for
high-throughput characterization of CHO cell banks. Our data show
that this method enables evaluation of a cryopreservation procedure
just 6 h after thaw.
Inventors: |
KAUFMANN; Hitto; (Ulm,
DE) ; FIEDER; Juergen; (Unterstadion, DE) ;
OTTO; Ralf; (Oggelshausen, DE) |
Correspondence
Address: |
MICHAEL P. MORRIS;BOEHRINGER INGELHEIM USA CORPORATION
900 RIDGEBURY ROAD, P. O. BOX 368
RIDGEFIELD
CT
06877-0368
US
|
Assignee: |
Boehringer Ingelheim Pharma GmbH
& Co. KG
Ingelheim
DE
|
Family ID: |
36118077 |
Appl. No.: |
12/491677 |
Filed: |
June 25, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11270236 |
Nov 9, 2005 |
7569339 |
|
|
12491677 |
|
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Current U.S.
Class: |
435/7.21 ;
435/29 |
Current CPC
Class: |
G01N 33/56966 20130101;
G01N 2333/4718 20130101 |
Class at
Publication: |
435/7.21 ;
435/29 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C12Q 1/02 20060101 C12Q001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 10, 2004 |
EP |
04026673.6 |
Claims
1-8. (canceled)
9. A method for establishing the vitality rate of a cryopreserved
CHO cell bank, the method comprising staining thawed CHO cells from
the cryopreserved CHO cell bank with Annexin V.
10. The method of claim 9, further comprising: quantifying the
number of thawed CHO cells that binds to Annexin V; quantifying the
number of thawed CHO cells that do not bind to Annexin V; and
calculating the ratio of Annexin V-binding thawed CHO cells to
Annexin V-non-binding thawed CHO cells.
11. The method of claim 10, wherein Annexin V is incubated with the
thawed CHO cells within 24 hours post-thaw.
12. The method of claim 10, wherein Annexin V is incubated with the
thawed CHO cells within 6 hours post-thaw.
13. The method of claim 10, wherein the Annexin V is labeled with
fluorescein isothiocyanate (FITC).
14. The method of claim 10, wherein the cryopreserved cell bank of
CHO cells comprises CHO cells that are cryopreserved into parts in
a liquid medium.
15. The method of claim 10, wherein the thawed CHO cells are thawed
from a portion of the cryopreserved cell bank.
16. The method of claim 10, wherein the thawed CHO cells are
cultured before being incubated with Annexin V.
17. (canceled)
18. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to the production of
biopharmaceuticals in CHO cells. Particularly, it pertains to the
generation of master-, working- and post-production cell banks of
high quality via cryopreservation. More particularly, it pertains
to the propagation and characterization of cells cryopreserved in
master-, working- and post-production cell banks. Furthermore, the
present invention refers to a novel strategy employing flow
cytometric (FC) analysis of Annexin V-stained cells for
high-throughput characterization of cryopreserved cell banks.
[0003] 2. Background of the Invention
[0004] The market for biopharmaceuticals for use in human therapy
continues to grow at a very high rate in the last decade. CHO cell
lines are one of the most attractive mammalian expression system
for production, safety, and regulatory aspects. To ensure
therapeutic products of uniform quality, the cell banking system of
these cell lines is crucial. Creation of Master Cell Banks (MCB),
Working Cell Banks (WCB), and Post Production Cell Banks (PPCB) of
CHO cells are essential steps in development of production
processes for biopharmaceuticals in that cell lines. The quality of
these banks is critical, as their generation not only supports
clinical development of the product but also ultimately the market
supply phase.
[0005] The main parameter that characterizes the quality of a cell
bank is the long term survival of cultured cells after thawing.
Moreover, besides the long term survival, robustness and stability
are also essential properties of a suitable cell bank. The time it
takes from thawing a vial to establishing inoculum cultures of
robust growth, genetic stability and high culture viability is
critical for assessing the quality of a cell bank. Finally, a cell
bank of high quality should guarantee for all of these parameters
to remain stable over a prolonged storage period of the bank. All
these characteristics highly depend on the method of
cryopreservation for a given production cell line.
[0006] Today, an increasing number of biopharmaceuticals is
produced from CHO cells due to their ability to correctly process
and modify human proteins. The first generation of CHO cell-based
production processes almost exclusively required the presence of
serum in the culture medium. Safety and regulatory benefits led to
development of new cell lines and culture regimes that now enable
serum-free cultivation of cells throughout the process (Merten,
1999). However, the removal of serum from the entire production
process also requires cells to be stored in master and working cell
banks with serum-free freezing media. A variety of strategies have
been described for cell banking of cells by using cryoprotectants
that are able to at least partially replace the protective effects
of serum (Groth et al., 1991). However, the success of any such
strategy highly depends on the cell line, the medium, and the
protocol for freezing and thawing. Therefore, evaluation of
different cryopreservation strategies is essential for successful
process development.
[0007] Currently, the first assessment of a newly generated cell
bank is performed by thawing a defined number of vials and
culturing cells for 5-10 passages. Cell number and viability as
determined by trypan blue exclusion are the routinely used
parameters to describe the recovery of cells after
cryopreservation.
[0008] Programmed cell death or apoptosis is a process crucial for
proper embryonic development and tissue homeostasis in the adult.
Programmed cell death is controlled by a specific subset of
molecules conserved in all multicellular organisms that converts a
death inducing signal into intracellular biochemical processes,
which ultimately lead to the complete destruction of the cell (Vaux
and Korsmeyer 1999). Once triggered, apoptosis proceeds, with
different kinetics depending on cell types, and culminates with
cell disruption and formation of apoptotic bodies. A critical stage
of apoptosis involves the acquisition of surface changes by dying
cells that eventually results in the recognition and the uptake of
these cells by phagocytes. Different changes on the surface of
apoptotic cells such as the expression of thrombospondin binding
sites, loss of sialic acid residues and exposure of phospholipids,
like phosphatidylserine (PS), were previously described.
Phospholipids are asymmetrically distributed between inner and
outer leaflets of the plasma membrane, with phosphatidylcholine and
sphingomyelin exposed on the external leaflet of the lipid bilayer
and phosphatidylserine predominantly observed on the inner surface
facing the cytosol. Cells undergoing apoptosis break up the
phospholipid asymmetry of their plasma membrane and expose PS,
which is translocated to the outer layer of the membrane. This
occurs in the early phases of apoptotic cell death during which the
cell membrane remains intact. PS exposure is, thus, an early and
wide-spread hallmark of dying cells. Annexin V, belonging to a
recently discovered family of proteins, the annexins, with
anticoagulant properties, has proven to be a useful tool in
detecting apoptotic cells, since it preferentially binds to
negatively charged phospholipids, like PS, in the presence of
Ca.sup.2+ and shows minimal binding to phosphatidylcholine and
sphingomyelin. Changes in PS asymmetry analyzed by measuring
Annexin V binding to the cell membrane were detected before
morphological changes associated with apoptosis occurred and before
membrane integrity was lost.
[0009] By conjugating FITC to Annexin V it is possible to identify
and quantify apoptotic cells on a single-cell basis by flow
cytometry (Steensma et al., 2003). Simultaneous staining of cells
with FITC-Annexin V (green fluorescence) and the non-vital dye
propidium iodide (red fluorescence) allows (bivariant analysis) the
discrimination of intact cells (FITC-PH-), early apoptotic
(FITC+PI-) and late apoptotic or necrotic cells (FITC+PI+).
SUMMARY OF THE INVENTION
[0010] As mention in the background section, quality of a
cryopreserved cell bank is critical in the use of a cell bank for
the production of biopharmaceuticals. In the meaning of this
invention, quality means, post thaw vitality, robustness,
phenotypic/genetic stability, and long term preservation quality of
cells, propagated and expanded starting from a cryopreserved cell
bank. The present invention is based on the surprising observation
that programmed cell death, particularly apoptosis, is the major
cause of cell death after thawing of cryopreserved CHO cells of a
frozen CHO cell bank. It also has been surprisingly demonstrated
that the evidence of early apoptosis in cells of a culture
propagated and expanded from a cryopreserved cell bank of CHO cells
correlates with the quality of the CHO cell bank, which means with
post-thaw vitality, robustness, phenotypic/genetic stability, and
long term preservation quality, observed when cells are propagated
and expanded starting from this cell bank. It has been furthermore
demonstrated that Annexin V is a suitable marker to detect early
apoptosis in CHO cells that have been thawed after
cryopreservation.
[0011] The present invention therefore pertains to the use of
Annexin V in characterizing a cryopreserved cell bank of CHO cells.
Particularly, the present invention relates to the use of Annexin V
in a process of characterizing a cryopreserved cell bank of CHO
cells, preferably shortly after thawing a portion of cells of such
a cell bank. According to a further embodiment of the present
invention, said process includes the steps: a) thawing a portion of
cells of a cryopreserved cell bank; b) cultivating said cells in a
culture medium; c) incubating said cells with Annexin V; d)
detecting cells and quantifying the number of cells that binds to
Annexin V; e) detecting cells and quantifying the number of cells
that do not bind to Annexin V; f) calculating the ratio of Annexin
V-binding versus Annexin V-non-binding cells.
[0012] According to a further embodiment, the present invention
also pertains to the use of Annexin V in a process of determining
the quality of a CHO cell bank, wherein said process includes the
steps: a) cryopreserving cells into parts in a liquid medium as a
cell bank; b) thawing a portion of the cryopreserved cells of said
cell bank; c) cultivating said cells in a culture medium; d)
establishing the vitality-rate of said thawed cells by staining
said cells with Annexin V. "Cryopreserving cells into parts" in a
liquid medium means, that each about 0.25 to 3E7 cells are frozen
in one to two ml of a liquid medium in a container. Per cell bank,
about 200 containers or vials are frozen.
[0013] The present invention also pertains to the use of a Kit
comprising Annexin V for the characterization of a cryopreserved
cell bank of CHO cells post thawing, wherein Annexin V is used to
determine the vitality-rate of the cryopreserved cells post
thawing. According to a preferred embodiment of the present
invention, the vitality-rate of cryopreserved cells post thawing is
determined by a process including the steps a) thawing a portion of
cells of a cryopreserved cell bank; b) cultivating said cells in a
culture medium; c) incubating said cells with Annexin V; d)
detecting cells and quantifying the number of cells that binds to
Annexin V; e) detecting cells and quantifying the number of cells
that do not bind to Annexin V; f) calculating the ratio of Annexin
V-binding versus Annexin V-non-binding cells. Furthermore, the
present invention also provides a Kit comprising Annexin V and a
package leaflet including the information to use Annexin V in
characterization of a cell bank of CHO cells by the inventive
process, described herein.
[0014] The present invention further provides a process of
characterizing a CHO cell bank including the steps a) thawing a
portion of cells of a cryopreserved cell bank; b) cultivating said
cells in an appropriate culture medium; c) incubating said cells
with Annexin V; d) detecting cells and quantifying the number of
cells that binds to Annexin V; e) detecting cells and quantifying
the number of cells that do not bind to Annexin V; f) calculating
the ratio of Annexin V-binding versus Annexin V-non-binding
cells.
[0015] According to a further embodiment, the present invention
also provides a process of measuring the vitality-rate of a CHO
cell bank post-thawing, including the steps a) thawing a portion of
cells of a cryopreserved cell bank b) cultivating said cells in an
appropriate culture medium c) incubating said cells with Annexin V;
d) detecting cells and quantifying the number of cells that binds
to Annexin V; e) detecting cells and quantifying the number of
cells that do not bind to Annexin V; f) calculating the ratio of
Annexin V-binding versus Annexin V-non-binding cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows a Fingerprint of cell death of CHO cell culture
post cryopreservation. CHO DG44 cells were frozen using a freezing
machine at a cell concentration of 2E7 cells/mL in 90% cultivation
medium and 10% DMSO. Cells were analyzed by FACS at indicated
timepoints. The first lane depicts forward scatter on the Y axis
and side scatter on the X axis. Gates were set to distinguish cells
that represent dead or living cells as judged by their morphology.
Lane 2 shows the same graphs and Annexin V positive CHO cells are
shown in white. Lane 3 depicts propidium iodide (PI) staining
plotted versus Annexin V staining. Dots are grey according to the
forward versus side scatter gating.
[0017] FIG. 2 shows 6 h post-thaw Annexin V staining predicts cell
bank quality. A. CHO DG44 cells were frozen using a freezing
machine at a cell concentration of 2E7 cells/mL in 90% cultivation
medium and 10% DMSO. The size of the cell bank was 220 vials. A)
FACS analysis of Annexin V stained cells 6 h post thaw. Data are
plotted as forward side scatter graph (gates as described for FIG.
1) and as Annexin V intensity plot. B) Growth and viability of
post-thaw cultures. Cells were counted for every passage.
Simultaneously, viability was determined by trypan blue
exclusion.
[0018] FIG. 3 shows different cell banks show different apoptosis
after thaw. CHO DG44 was frozen using a freezing machine with a
cell concentration of 1E7 or 2E7 cells/mL in different freezing
media. The size of the cell banks was >200 vials. Cells were
cryopreserved using a freezing machine or a polystyrene box in two
different freezing media. Cultures were analyzed by Annexin V
staining 6 h post thaw.
[0019] FIG. 4 shows use of early apoptosis measurements for testing
of medium quality. CHO DG44 cells were frozen using a freezing
machine at a cell conc. of 2E7 cells/mL in 90% cultivation medium
and 10% DMSO. The size of the cell bank was 220 vials. Different
media preparations were used for generation of three cell banks.
Cultures were analyzed by FACS 6 h post thaw for cell cycle
distribution, forward-side scatter, and Annexin V staining.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The term "cell culture(s) was (were) initiated" refers to
the time point, when cryopreserved cells are thawed and transferred
to culture conditions suitable for the propagation and grown up of
said cells to a cell culture.
[0021] The term "cell culture" means multiple cells cultivated in
one container under conditions suitable for the growth of the
cells.
[0022] The term "post thaw vitality" shall mean the time-course of
post thaw recovery of cells propagated from a given cell bank
measured in the percentage of viable cell to non-viable cells at
the time of sub-cultivation. This parameter is essential for the
time it takes to establish robust inoculum cultures and subsequent
scale up for large scale cultivation. It therefore is a critical
process parameter,
[0023] The term "robustness" describes the reproducibility with
which cell cultures recover after cryopreservation according to an
established post thaw vitality pattern. A robust cell bank would
result in minor differences amongst the post thaw performances of
its different vials. The term "vial/vials" means a number of cells
frozen in one container. Normally, a vial contains about 0.25 to
3E7 cells in one to two ml of a liquid medium.
[0024] The term "phenotypic/genetic stability" of cells is defined
by the change in RNA and protein expression level observed when
cells from a given cell bank are cultivated over a period of time
relevant to the given process format, e.g., for about 100 to 300
sub-passages. A cell bank of poor quality, meaning low post-thaw
vitalities could give rise to poor phenotypic/genetic stability due
to a high selection pressure when cultures are initiated. A low
post-thaw vitality in the meaning of the present invention means
that not more than 50%, preferably not more than 30%, more
preferably not more than 20%, and furthermore preferably not more
than 15% of the cells are Annexin V positive within 6 h post thaw.
In other words, a high quality cell bank is characterized in a
vitality-rate post thawing of more than 50%, preferably more than
70%, more preferably more than 80%, and furthermore preferably more
than 85%, which means Annexin V negative within 6 h post thaw.
[0025] The term "long term preservation quality" is defined by the
change of the three above described parameters over a prolonged
period of cryopreservation, for example about up to 20 years, of a
given cell bank.
[0026] The necessity to remove serum from mammalian cell culture
processes, particularly from culturing CHO cells, has resulted in
higher sensitivity of cultures towards cell death at different
stages of a production process. A detailed analysis of cell death
during thawing of CHO cells subsequent to cryopreservation was
provided. It was a surprisingly finding of the present invention
that programmed cell death or apoptosis is the predominant form of
cell death caused by cryopreservation of CHO cells. As early as 3 h
hours after, an increased level of phosphatidylserine was
surprisingly observed on the cell surface of CHO cells. Display of
phosphatidylserine on the surface of CHO cells as a result from
loss of phospholipids asymmetry is an early hallmark of programmed
cell death. At this point, cellular membrane integrity is still
intact.
[0027] Early time points after thaw, e.g., 3 h to 24 h post
thawing, showed Annexin V positive stained CHO cells to still be
morphologically intact (as seen in forward-side-scatter plots). At
later time points (around 24-48 h post thaw), the majority of
Annexin V positive stained CHO cells appeared to show signs of
shrinking/membrane disassembly as seen by forward-side-scatter
analysis. In accordance with these surprisingly findings, the
percentage of CHO cells that could be stained with propidium iodide
(PI) only increased significantly 24 h post thaw. These data
describe for the first time nature and time course of cell death of
CHO cells post cryopreservation. Certain CHO cells show signs of
having initiated a destruction program within a few hours after
being placed in warm culture medium and subsequently these cells
disassemble over the period of 48 h.
[0028] Annexin V was initially described as a vascular protein with
strong anticoagulant properties (Reutelsberger et al., 1985). It
appears to belong to a multigene family of proteins defined by a
repeated motif, originally termed the endonexin loop. In the
meantime, gene sequences coding for the Annexin V protein of
several species have been identified and cloned. For example, see
NCBI Protein Data Base Accession No. NP.sub.--01145.
[0029] The present invention therefore relates to the use of
Annexin V in characterizing a cryopreserved CHO cell bank.
Particularly, the present invention relates to the use of Annexin V
in a process of characterizing a cryopreserved cell bank of CHO
cells. According to a further embodiment of the present invention,
said process includes the steps: a) thawing a portion of cells of a
cryopreserved cell bank; b) cultivating said cells in a culture
medium; c) incubating said cells with Annexin V; d) detecting cells
and quantifying the number of cells that binds to Annexin V; e)
detecting cells and quantifying the number of cells that do not
bind to Annexin V; and f) calculating the ratio of Annexin
V-binding versus Annexin V-non-binding cells.
[0030] According to a further embodiment the present invention also
pertains to the use of Annexin V in a process of determining the
quality of a CHO cell bank, wherein said process includes the
steps: a) cryopreserving cells into parts in a liquid medium as a
cell bank; b) thawing a portion of the cryopreserved cells of said
cell bank; c) cultivating said cells in a culture medium; and d)
establishing the vitality-rate of said thawed cells by staining
said cells with Annexin V. Criteria for the quality of a CHO cell
bank are post-thaw vitality, robustness, phenotypic/genetic
stability, and long term preservation quality, observed when cells
are propagated and expanded starting from this cell bank. These
criteria are mainly affected by the number of CHO cells which
survive the freeze/thaw process, in other words the number of
"intact cells." A high number of intact CHO cells guarantee a fast
growth of the initial cell culture, reduce the number of
sub-cultivation steps necessary to expand the cell culture up to
the large fermentation scale and therefore to minimize the risk
that genetic variants may be established and/or dominated the
culture. The vitality-rate is preferably estimated by Annexin V
staining, which means the detection and quantification of Annexin
V-binding cells. Therefore, according to a more preferred
embodiment of the present invention, the process of the Annexin V
staining in order to estimate the vitality-rate of CHO cells
propagated and expanded after thawing includes the steps: a)
incubating the post-thawed cells with Annexin V; b) detecting cells
and quantifying the number of cells that binds to Annexin V; c)
detecting cells and quantifying the number of cells that do not
bind to Annexin V; and d) calculating the ratio of Annexin V
binding vs. Annexin V non-binding cells. Examples of CHO cells are
given in Table 1.
TABLE-US-00001 TABLE 1 CHO cell lines CHO Cell line Order Number
CHO ECACC No. 8505302 CHO-K1 ATCC CCL-61 CHO-DUKX ATCC CRL-9096
(=CHO duk-, CHO/dhfr-) CHO-DUKX B1 ATCC CRL-9010 CHO-DG44 Urlaub et
al., Cell 33[2], 405-412, 1983 CHO Pro-5 ATCC CRL-1781
[0031] A variety of assays for detection of apoptotic cell cultures
has been described, including DNA-laddering and TUNEL assays
(Gavrieli et al., et al., 1992; Wijsman et al., 1993). However,
flow cytometric methods of apoptosis detection offer several
advantages over the above techniques as they allow rapid
quantification of properties of thousands of cells. The use of
flow-cytometric measurements allow rapid high throughput evaluation
of different strategies do generate a successful cell bank for a
given cell line. For this reason and according to a further
embodiment of the present invention, the Annexin V is labeled with
fluorescein isothiocyanate (FITC). This allows the use of a
flow-cytometric Annexin V affinity assays to estimate the
vitality-rate of cells post thawing. Therefore, the present
invention also pertains to a process of staining cryopreserved
cells of a CHO cell bank post thawing a portion of cells of that
cell bank with FITC-labeled Annexin V in a flow-cytometric assay.
An example of such an assay is given herein more in detail in the
section Examples.
[0032] The results provided herein gives an explanation for results
typically seen when a freeze/thaw step is monitored by trypan blue
exclusion analysis of cell viability (standard method which reflect
the state of the art in characterization of cell banks). These
classic thaw-control experiments generally show a high number of
viable cells right after thaw and viability only decreases between
24 h and 48 h post thaw. A broad analysis of data obtained for CHO
cell banks surprisingly revealed the predictive value of
flow-cytometric Annexin V affinity assays. The percentage of
apoptotic CHO cells measured as early as 6 h post thaw provides
information on the success of a cryopreservation strategy that is
usually only gained in classic thaw control experiments over a
period of 2-10 days. The present invention therefore also provide
the use of Annexin V in characterization of a CHO cell bank or in a
process of determining the quality of a CHO cell bank, wherein the
incubation step with Annexin V is performed within 24 h,
preferably, 18 h, more preferred 12 h, further more preferred 9 h,
further more preferred 6 h post-thaw and start of cultivation of
the cryopreserved cells. In this connection use of flow-cytometric
assay using FITC labeled Annexin V is the most preferred
method.
[0033] According to a further embodiment, the present invention
also pertains to the use of a Kit comprising Annexin V for the
characterization of a cryopreserved CHO cell bank post thawing,
wherein Annexin V is used to determine the vitality-rate of the
cryopreserved CHO cells post thawing. According to a preferred
embodiment of the present invention, the vitality-rate of a cell
culture is determined by a process including the steps: a) thawing
a portion of cells of a cryopreserved cell bank; b) cultivating
said cells in a culture medium; c) incubating said cells with
Annexin V; d) detecting cells and quantifying the number of cells
that binds to Annexin V; e) detecting cells and quantifying the
number of cells that do not bind to Annexin V; and f) calculating
the ratio of Annexin V-binding vs. Annexin V-non-binding cells.
Furthermore, the present invention also provides a Kit comprising
Annexin V and a package leaflet including the information to use
Annexin V in characterization of a CHO cell bank.
[0034] The present invention further provides a process of
characterizing a CHO cell bank including the steps: a) thawing a
portion of cells of a cryopreserved cell bank; b) cultivating said
cells in an appropriate culture medium; c) incubating said cells
with Annexin V; d) detecting cells and quantifying the number of
cells that binds to Annexin V; e) detecting cells and quantifying
the number of cells that do not bind to Annexin V; and f)
calculating the ratio of Annexin V-binding versus Annexin
V-non-binding cells. According to a further embodiment, the present
invention also provides a process of measuring the vitality-rate of
a CHO cell bank post-thaw, including the steps: a) thawing a
portion of cells of a cryopreserved cell bank b) cultivating said
cells in an appropriate culture medium c) incubating said cells
with Annexin V; d) detecting cells and quantifying the number of
cells that binds to Annexin V; e) detecting cells and quantifying
the number of cells that do not bind to Annexin V; and f)
calculating the ratio of Annexin V-binding vs. Annexin
V-non-binding cells. As shown by the present invention, the
percentage of apoptotic CHO cells measured as early as 24 to 6 h
post thaw provides information on the success of a cryopreservation
strategy for CHO cells that is usually only gained in classic thaw
control experiments over a period of 2-10 days. The present
invention therefore also provide the processes of characterizing a
CHO cell bank or measuring the vitality-rate of a cell bank
post-thaw, wherein the incubation step with Annexin V is performed
within 6-24 hours post-thaw and start of cultivation of the
cryopreserved cells.
[0035] CHO cells can be successfully stored in liquid nitrogen at
around -196.degree. C. for prolonged periods of time. However,
cells can usually not be frozen and thawed without the addition of
cryoprotectants such as DMSO. Even then, the freeze-thaw process
will result in the loss of cells. A critical parameter for the
amount of this loss is the protocol used for the freeze and the
thaw process. Generally it is beneficial to control the temperature
decrease during freezing to ensure that the temperature drops at a
rate to of around one degree per minute. To thaw CHO cells
successfully it is required to warm the cryovial (thawed cells) as
fast as possible to 37.degree. C., the optimal cultivation
temperature of almost all CHO cell lines. One way to ensure high
recovery rates of cultures post thaw is to freeze cells in a medium
containing high amounts of serum.
[0036] Recently, much work was focused on developing
cryopreservation strategies for CHO cells without the use of serum
in the freezing medium. A variety of cryoprotectants can facilitate
successful long-term storage of cells without the presence of
serum. In addition, the composition of the basal medium as such has
a significant impact on the success of any cryopreservation. The
described fast-track quality assessment of any such strategy will
allow drastically reduced development times that are needed to
generate reliable protocols for generation of a master or working
cell bank for new CHO production cell lines. According to a further
embodiment, the present invention also provides a process of
analysing a culture medium for CHO cells with regard to the use of
said medium for cryopreservation of CHO cells including the steps:
a) cryopreserving CHO cells in a culture medium suitable for
culturing those cells, preferably at temperatures below
-100.degree. C., e.g., in fluid nitrogen, b) thawing a portion or
all of the cryopreserved cells; c) cultivating said cells in an
appropriate culture medium; d) incubating said cells with Annexin
V; e) detecting cells and quantifying the number of cells that
binds to Annexin V; f) detecting cells and quantifying the number
of cells that do not bind to Annexin V; and g) calculating the
ratio of Annexin V-binding versus Annexin V-non-binding cells.
Suitable culture media are those based on commercially available
media such as Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640
(Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), Minimal
Essential Medium (MEM; Sigma), Iscove's Modified Dulbecco's Medium
(IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, Calif.),
CHO-S-Invtirogen), serum-free CHO Medium (Sigma), and protein-free
CHO Medium (Sigma). According to a preferred process said medium
includes specific cryprotectants such as DMSO, BSA,
methylcellulose, or glycine. According to a more preferred
embodiment of this process, the incubation step with Annexin V is
performed within 6-24 hours post-thaw and start of cultivation of
the cryopreserved cells. Use of FITC-labeled Annexin V in
flow-cytometric assay is a furthermore preferred embodiment of the
inventive process.
[0037] The invention generally described above will be more readily
understood by reference to the following examples, which are hereby
included merely for the purpose of illustration of certain
embodiments of the present invention and are not intended to limit
the invention in any way.
Abbreviations:
[0038] BSA: bovine serum albumin CHO: Chinese hamster ovary DMSO:
dimethyl sulfoxide ELISA: enzyme-linked immunosorbant assay FACS:
fluorescence activated cell sorter FITC: fluorescein isothiocyanate
MCB: master cell bank PBS: phosphate buffered saline PCR:
polymerase chain reaction PI: propidium iodide PPCB: post
production cell bank PS: phosphatidylserine WCB: working cell
bank
Methods:
Cell Culture:
[0039] Cell banks of several CHO suspension cell lines used for
production and development of biopharmaceuticals were investigated.
All cell lines used are proprietary and their protein product may
not be revealed. All cell lines used at production and development
scale were maintained in serial seedstock cultures in
surface-aerated T-Flasks (Nunc, Denmark) in incubators (Thermo,
Germany) or sparged spinner flasks (Wheaton, USA) in specially
designed incubator rooms at a temperature of 37.degree. C. and an
appropriate mixture of air and 5% CO.sub.2. Seedstock cultures were
split every 2-3 days with an appropriate splitting ratio and seed
density. Cell concentration was determined in all cultures by using
a hemocytometer. Viability was assessed by the trypan blue
exclusion method. The cultures originated from master, working or
safety cell banks and were thoroughly tested for sterility,
mycoplasma, and the presence of adventitious agents. All operations
took place in air-filtered laboratories and under strict procedures
complying with "current Good Manufacturing Practices (cGMP). All
culture media used are proprietary and their composition may not be
revealed.
Cryopreservation:
[0040] Freezing and thawing were performed according to standard
protocols and the fundamental law "freezing low-thawing fast." The
cultures were taken in exponential phase from suspension cultures
and were frozen in 1.8 mL plastic vials (Nunc, Demark) either by a
computer-controlled freezing machine (Consarctic, Germany) with a
specially designed freezing program or by a Styrofoam box in a
-70.degree. C. freezer. After freezing to -100.degree. C. (freezing
machine) or -70.degree. C. (Styrofoam box) the cells were
transferred to liquid nitrogen containers. Vials were stored in the
gas phase of the liquid nitrogen (<-150.degree. C.). The cell
concentration in the freezing vials was 1-3E7 cells. DMSO (Merck,
Germany) was used as a cryoprotectant at a concentration of 10%.
The detailed freezing media used are also proprietary and their
composition may not be revealed. CHO cells were thawed in an
37.degree. C. water bath, diluted with appropriate medium, and
centrifuged (Thermo, France) at 180.times.g. Subsequently cells
were seeded in cultivation medium with a defined cell
concentration.
Flow Cytometry:
[0041] Green Annexin-V-FITC and red PI fluorescence were measured
with a Coulter Epics XL flow cytometer (Coulter, Germany) using
Expo32 software. Excitation was elicited at 488 nm with an Argon
laser and measured using the standard band pass (530.+-.20 nm) and
long pass (>570 nm) filters. In each sample, 10000 events for
apoptosis and 3000 events for cell cycle analysis were measured.
Data were analyzed with the Expo32 analysis tool. To discriminate
between dead and apoptotic cells, the membrane-impermeable DNA
stain propidium iodide (PI) was added in parallel to Annexin V to
the cell suspension. With this double staining it is possible to
discriminate between vital, early-apoptotic, late-apoptotic, and
necrotic cells. The Annexin V assay was performed according to the
vendor's protocol (Becton-Dickinson, Germany). The cells were
analyzed and apoptosis was quantified by flow cytometry. Propidium
iodide staining was further used to determine cell cycle
distribution. Samples were taken from cell suspensions without
fixation. Cultures were centrifuged and washed with PBS (Gibco,
Germany). Then, the PI staining solution (BioSure, USA) was added
and incubation proceeded for 30 min. Subsequently, cells were
analyzed by flow cytometry. The proportion of cells in each phase
of the cell cycle was obtained through the Multicycle program
(Phoenix Flow, USA).
Results:
[0042] Quantitative analysis of cell death during the thaw of
cryopreserved CHO cells:
[0043] Although it is known that CHO cells die when cultures of
cells are reinitiated after cryopreservation the kinetic and nature
of this cell death have not been described in great detail. To
quantify the extent of programmed cell death in cell cultures of
CHO cells during the first hours after thawing we determined the
amount of phosphatidylserine displayed on the outer membrane of CHO
cells. CHO DG44 cells and derivatives of this cell line are widely
used for production of biopharmaceuticals. We first analyzed a cell
bank of 220 vials generated for a CHO-derived production cell line.
The cell bank was generated with a freezing medium free of animal
components, such as serum, and was known to be particularly
sensitive with regard to culture viability after thawing. Cells
were analyzed by double staining with Annexin V and propidium
iodide at time points Oh, 3 h, 6 h, 24 h, and 48 h. A high
percentage of approximately 30% Annexin V positive cells was
detected right after cells were placed in cultivation medium (Oh),
increasing to almost 50% within the first 3 h, and remaining at
above 40% for the first 48 h post thaw. For all CHO DG44 cell banks
analyzed so far, the peak level of Annexin V positive cells was
reached between 3 h and 24 h post thaw. In contrast to that, the
amount of PI positive cells remained relatively low immediately
after thaw before it increased markedly 24 h post thaw. In the
initial phase of apoptosis, PS is displayed on the cell surface and
Annexin V binds it target. In this early phase, the plasma membrane
is still able to exclude PI and, thus, cells are Annexin V+/PI-
(FIG. 1, lower right quadrant). Subsequently, cells loose their
plasma membrane integrity and their ability to exclude PI. These
late apoptotic cells are Annexin V+/PI+ (FIG. 1, upper right
quadrant). Therefore, these experiments demonstrate that the
transition from early to late stage apoptosis only occurs after the
first 6 hours post thaw. This is also seen in the forward-side
scatter diagram (FIG. 1), showing an increase in very small cells,
representing destructed cell bodies (FIG. 1) at the transition
point around 24 hours post thaw.
Post-Thaw Analysis by Annexin V Staining Serves as Predictive Early
Marker for CHO Cell Bank Quality:
[0044] As quantification of apoptosis by Annexin V staining can be
performed rapidly for a high number of samples it offers an
attractive method for high-throughput analysis of different
cryopreservation methods. We analyzed two CHO cell banks of two
different CHO production cells by Annexin V staining and compared
these data to trypan blue exclusion staining measured for six
passages in culture (standard thaw control). As seen in FIG. 2A,
the amount of phosphatidylserine-displaying cells varied
significantly between cultures thawed from the two different cell
banks (35% in thaw 1 versus 67% in thaw 2). The classic thawing
control (FIG. 2B) showed the same significant difference between
the culture viabilities from the different banks. Thaw 1
demonstrated a drop to 83% before the culture recovered to a
viability of more than 90% four days post thaw. In contrast to
that, only 60% of cells were viable 24 h after thaw 2. This culture
took eleven days to reach 90% viability as determined by trypan
blue exclusion. A cell bank of this quality would clearly not be
suitable for a commercial production process. These data
surprisingly demonstrate that the percentage of cells displaying PS
on their surface predicts the outcome of classic thawing controls
as early as 6 h post thaw.
Cell Bank Quality can Vary Dramatically Amongst Different Cell
Lines, Cryopreservation Protocols, and Quality of Cultivation
Medium:
[0045] To test the newly described method for cell bank quality
assessment we analyzed a number of cell banks. Two CHO DG44 cell
banks were generated, one containing 0.8% BSA in the freezing
medium, the other containing a 50:50 mixture of conditioned and
fresh medium without BSA. As depicted in FIG. 3, the cells showed
significantly lower rates of early apoptosis for both freezing
media. Furthermore, a beneficial effect of adding 0.8% BSA compared
to storing cells in conditioned medium was only evident when
styrofoam boxes were used.
Use of Early Post-Thaw Apoptosis Measurements for Fast-Track
Development Strategies:
[0046] As time to clinic and market supply is a crucial parameter
for successful development of new biopharmaceuticals there is a
growing interest in fast-track screening methods for process
optimization and process improvements. We therefore determined the
feasibility of using Annexin V measurements just 6 h post thaw to
evaluate strategies for establishing successful cryopreservation of
new CHO production cell lines.
[0047] We surprisingly find out that early post-thaw apoptosis
measurements are a sensitive tool for assessment of raw materials
quality. An essential factor for successful cryopreservation of CHO
cells is the quality of the medium used. Generally, GMP includes
clearly defined expiry dates for use of media for culturing CHO
cells. We investigated whether the harsh conditions of a
freeze-thaw process may result in shorter timeframes for the use of
media compared to standard cultivation procedures. We used a medium
with a shelf life of four weeks for cultivation of CHO cells to
investigate this further. FIG. 4 shows early apoptosis data (6 h
post thaw) for CHO cells that were cryopreserved in freshly
prepared medium (4 bottom), medium that was stored at 4 C for four
weeks (4 top) and medium that was stored at 4 C for four weeks and
was supplemented with a fresh preparation of an essential compound
immediately before use (4 middle). These data show a decreased thaw
performance for cells banks generated with this medium just before
its expiration date. This effect could be reduced significantly by
adding a newly prepared essential component of the relevant medium.
Simultaneous determination of the cell cycle distribution 6 h post
thaw showed that four week old medium had no growth inhibitory
effect. These results surprisingly demonstrate how early post-thaw
apoptosis measurements could be used for sensitive analyses of
media and their components with regard to the use of media
preparations for cryopreservation of CHO cells.
* * * * *