Cell Line Development and Engineering
Workshop, Prague, March 2008
Important Topics in the Expression of Recombinant
Antibodies from CHO Cells
Robert Young/Lonza Biologics/Mar 2008
Disclaimer
„
Certain matters discussed in this presentation may constitute
forward-looking statements. These statements are based on
current expectations and estimates of Lonza Group Ltd, although
Lonza Group Ltd can give no assurance that these expectations
and estimates will be achieved.
„
The actual results may differ materially in the future from the
forward-looking statements included in this presentation due to
various factors. Furthermore, Lonza Group Ltd has no obligation to
update the statements contained in this presentation.
„
Note: All slides are incomplete without verbal comments.
slide 2
Talk Outline
„
Introduction
„
Transcription and Expression Vector Technology
„
Transcription: Lonza’s Experience
„
Enhancing translation
„
Enhancing secretion
„
Antibody isotype and expression levels
„
Summary
slide 3
Introduction
„
Achieving high antibody concentrations is the combination of many
factors including:
„ Expression vector technology.
„ Cell line.
„ Media and feeds.
„ Process.
„
In order to improve the concentrations of recombinant protein
achievable in a cell culture process, all aspects of the production
process require optimisation.
„
New technologies are emerging that can be applied to recombinant
protein production.
„ Focus on the application of new technologies to a commercially
relevant expression platform.
slide 4
The Question
„
How can the steps involved in the central dogma of protein
expression be manipulated to improve production of recombinant
antibodies?
„
From Gene to Protein
„
Transcription
„
Translation
„
Secretion
slide 5
Transcription and Expression Vector Technology
Key Features of mammalian expression Vectors
(Ludwig 2006 Bioproc Int Supp. 4:14-23)
slide 7
The importance of the ‘position effect’
„
The level of transcription depends on the site of transgene
integration
„ Integration may occur in open chromatin or in a region of
condensed inactive chromatin
„ Most genomic sites repress transgene expression (in
humans, 60 % of sites are silent)
„ Extensive screening of clones required to isolate rare events
of integration in active DNA
„
Sequence elements have been used to reduce influence of the
“position” effect
„ For example; Insulators, MAR/SAR and STAR etc
slide 8
Other approaches to improving transcription
„
„
„
„
New or enhanced selectable markers (e.g. stringency)
Stronger promoters (e.g. murine CMV vs human CMV)
Other promoters/transcription units (e.g., CHEF1)
Different vector approaches (e.g. retroviral vectors, artificial
chromosomes, etc)
slide 9
Insulator elements (1)
(West and Fraser 2005 Hum Mol Genet. 14:R101-R111.)
„
Insulators can be defined as:
„ Sequence elements that possess a common ability to protect
genes from inappropriate signals emanating from their
surrounding environment (phenotypic definition)
„
5’ HS4 element from chicken β-globin locus has blocking and barrier
function:
„ Prevents the action of gene enhancers when placed between
enhancer and promoter sequence (blocking)
„ Halt the progression of chromosome silencing, hence the
formation of heterochromatin (barrier)
„ Footprint IV sequence binds USF protein leading to recruitment
of enzymes responsible for histone H3/H4 protein acetylation and
H3K4 methylation
„ Prevents encroachment of H3K9 methylation and chromatin
condensation
slide 10
Insulator elements (2)
Usage
Recillas-Targa 2002 Proc Natl Acad Sci (USA) 99:6883-88
http://www.gla.ac.uk/cancerpathology/genemech/awest/using_insulators.PDF
„
„
Flanking a transgene with two copies of an insulator can shield it from
chromosomal position effect following stable integration
The majority of chromosomal loci are transcriptional silent in mature
mammalian tissues so consequently position effect manifests as
unwanted transcriptional silencing
slide 11
Stabilising and anti-repressor element (STAR)
(Kwaks et al. 2003 Nat. Biotech. 21, 553-58)
„
STAR elements were
isolated using a genetic
screen
„
STAR element properties
„ Transcription blocker
„ Increase expression from the hCMV promoter
„ Increase the number of colonies that expressed the transgene
„ Stable expression of SEAP over 60 generations
„ STAR-SelectTM leads to 6x higher MAb yields and 35-50 pg/cell/day
with picking only 24 colonies (before process optimization)
„
Stringency increased by linking transgene to zeomycin via an IRES
slide 12
Expression Augmenting Sequence Element
(EASE) vectors (Amgen)
(Aldrich et al. 2003 Biotechnol Prog. 19:1433-38)
„
Isolated from CHO cells proximal to a unique integration site of a
recombinant TNF-Fc fusion protein (3.6 kb)
„
One EASE element per transcription unit on separate light (neo) and heavy
chain (dhfr) expression vectors were used. In each case an IRES linked the
Ig gene to the relevant selectable marker
„
Not a promoter/enhancer/MAR/SAR/LCR element and has no ORF so does
not encode a transactivator
„
Cloning of CS-9 (CHO DXB-11 serum-free suspension adapted variant)
transfectants in 50 nM MTX-containing media
„ EASE clones produced 15-20 pcd (85-95% viability) and were stable
over 20 generations
„ Non-EASE clones produced 1.5-2.0 pcd (28-41% viability).
„
Conclusion was that EASE-vectors allowed for greater co-amplification of the
antibody heavy and light chains with increased concentrations of MTX in the
growth medium. Viability and stability were both enhanced.
„
Mechanism of action is unknown, but it might be to facilitate integration into
the host genome
slide 13
Matrix Attachment Regions (MARs)
„
AT-rich DNA elements that bind to the nuclear matrix. Not all AT rich
regions are MARs. Not all MAR sub-fragments that bind nuclear matrix
have the ability to enhance and insulate transgene expression (Phi-Van
and Strätling 1996 Biochemistry 35, 10735-42)
„
Classical MARs are located near the boundaries of active chromatin
domains and may map with loop anchorage points (5’ boundary of
chicken lysozyme locus; Phi-Van and Strätling, 1988 EMBO J. 7:655-64)
„
Often contain topoisomerase consensus sequences
„
Their action in functional transgenic assays define them as classical
insulators
„
MARs are thought to be involved in creating high-order structuring of
chromatin in topologically independent loop domains (open chromatin)
„
Not much is known about how MARs work at the molecular level and
why they can have beneficial effects on transgene expression
„
They are relatively small (=<3 kb) and can be incorporated into vectors
easily
slide 14
CHEF1 Vectors (ICOS)
(Running Deer et al. 2004 Biotechnol Prog. 20, 880-9)
„
12Kb 5’ and 4 kb 3’ flanking sequences
of a highly expressed CHO gene
(CHEF1α) with EF-1α promoter
„
Average pooled transfectant productivity
with a Fc Fusion (DG44) was 6 to 35fold higher than pCDNA3 or pEF1/V5
(human E1Fα promoter). Top clone
produced ~130 mg/L
„
CHEF1-driven expression of SEAP or
CCR4 was 13 to 280-fold and 10-fold
greater, respectively, than pCDNA3 or
pEF1/V5 in non-CHO lines
„
MTX not required for stability
slide 15
Stringency of selection – NPT engineering
(Sautter et al. 2004 Biotech Bioeng. 89, 530-8)
„
„
„
„
„
Incorporated into the commercial BI-HEX expression
system (Boehringer-Ingelheim)
Based on impairment of the activity of the selectable
marker enzyme neomycin-phosphotransferase (NPT)
The ratio of high producing cells to total was higher with the
mutant NPT marker compared to the wild type
With this system, higher levels of mutant NPT mRNA,
higher specific productivities and titres were achieved.
Elevated productivities were ascribed to higher gene copy
numbers, integration into sites of high transcriptional
activity or a combination of both
slide 16
Transcription: Lonza’s Experience
Typical GS Vector for Antibody Expression
„
„
„
Gene of interest driven by
strong promoter
„ hCMV-MIE
Light Chain Gene
Intron A
GS is inhibited by methionine
sulphoximine
GS cDNA driven by weak
promoter
„ SV40E
SV40 Poly-A
hCMV-MIE promoter
5'UTR
5'UTR
5'UTR
Intron A
5'UTR
hCMV-MIE promoter
GS Vector
Heavy Chain Gene
11445 bp
SV40 intron + Poly-A
„
Increase selection stringency
„ Weak promoter on GS
gene - selects for rare
integration into
transcriptionally efficient
sites in genome
SV40 Poly-A
GS cDNA
SV40E (and SV40 ori)
pEE6 ori
beta-lactamase
slide 18
Quantification of mRNA levels
„
Heavy Chain
Light Chain
1
1
Relative Light Chain
„
As the site of integration into the genome is likely to have a major effect
on transcription (position effect), a panel of cell lines expressing a model
antibody with a variety of productivities was evaluated.
Relative mRNA levels of the heavy and light chain were determined by
quantitative RT-PCR.
Higher levels of mRNA were associated with higher product
concentrations.
Relative Heavy Chain
„
0
Col 1 vs Col 2
Plot 1 Regr
0
0
200
400
600
800
1000
1200
1400
1600
Antibody concentration (mg/L)
1800
2000
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Antibody concentration (mg/L)
slide 19
Alternative to the hCMV-MIE Promoter
„
The murine CMV-MIE (mCMV-MIE) promoter has been reported to
be stronger than the human alternative (hCMV-MIE)
„
„
(Addison et al., 1997 J.Gen.Vir. 78:1653-61, Rotodara et al., 1996
Gene 168: 195-8)
To test this, GS expression vectors were constructed to enable
expression of GFP using either the hCMV-MIE promoter plus the
hCMV-MIE Intron A (as present in the GS vectors) or the mCMVMIE promoter.
„
„
„
„
Transfect CHO-K1 cells, plate into T175 flasks.
Select with MSX for 26 days.
Analyse pool of transfectants for fluorescence at 510 nm
(excitation 488 nm) with Coulter Elite flow cytometer.
Record mean channel number for the highest 10% of the
viable cell population.
slide 20
Alternative to the hCMV-MIE Promoter
Use of the mCMV-MIE promoter resulted in increased
mean fluorescence indicating increased protein expression
is possible using this promoter.
350
300
Mean Channel Number
„
250
200
150
100
50
0
Control
cells
hCMV +
hCMV intron
mCMV
slide 21
Alternative Promoters
„
Do the observed increases in GFP expression levels predict
increased levels of a more complex secreted protein such as an
antibody?
„
Evaluated GS vectors encoding a model antibody utilising
„
the hCMV-MIE promoter and human Intron A.
„
the mCMV-MIE promoter.
„
the mCMV-MIE promoter and human Intron A.
slide 22
Evaluating Vector Improvements
„
Method for evaluating vector improvements.
Transfect host cells with vector
Identify single colonies per
3-4 weeks
well
100 transfectants
Transfer to 24 well plate
100 transfectants
2 weeks
100 data points
Productivity assessment
(quantitative)
Compare against control
slide 23
Antibody Expression
using the mCMV-MIE Promoter
„
Use of the mCMV-MIE promoter plus hCMV-MIE Intron A resulted
in equivalent mean antibody concentrations to the hCMV-MIE
promoter plus hCMV-MIE Intron A.
„
Use of mCMV-MIE promoter alone resulted in reduced mean
antibody concentrations.
Antibody
concentration (mg/L)
250
200
*
150
100
50
0
mCMV
mCMV +
hCMV intron
hCMV +
hCMV intron
*
P < 0.05 vs
hCMV/intron
slide 24
Lonza’s experience of MARs in the GS Expression
System (1)
„
MAR element used:
„ 3 kb chicken lysozyme
MAR element (Phi-Van
et al. 1990 Mol Cell
Biol. 10, 2302-07).
„
Mid
5’
MAR element placed in
3 different locations
within expression
vector.
3’
slide 25
Lonza’s experience of MARs in the GS Expression
System (2)
slide 26
Why MARs weren’t successful in the GS
Expression System? Possible answers
„
„
„
„
„
GS system biases integration into sites of highest transcriptional
activity, hence masking the benefit of MAR elements
The 5’ chicken lysozyme MAR may not necessarily be the best
choice
The human β-globin MAR was found to be better in a recent
screen of MARs. Used to simplify DHFR-based amplification/cell
line selection (Kim et al. 2004 J Biotechnol. 107, 95-105)
Latterly reported that the human β-interferon SAR (scaffold
attachment region) was even more effective when 2 copies were
used in the 5’ and 3’ flanking regions (Kim et al. 2005 21:933-37)
Selexsis have better understanding of MAR and how to use them
and can get CHO cell lines with Qp of up to 110 pcd (up to 65pcd
from GS-CHO)
slide 27
Conclusions
„
High levels of transcription are important for maximal antibody
productivity.
„ Increasing levels of transcription is important to move from low
to high producing cell lines.
„
Alternative promoters can result in increased protein expression.
„ However, this effect was not observed when the stronger
promoter was used to express a complex secreted protein.
„
What steps downstream of transcription can be manipulated to
increase productivity?
„ Can optimisation of these steps increase the amount of protein
expressed from highly productive cell lines further.
slide 28
Enhancing Translation
Gene Optimisation
„
Gene optimisation aims to improve productivity of a given protein
by altering the gene sequence without altering the protein it
encodes.
„
Codon optimisation
„ Use of optimal codons for the desired host cell.
„
Optimisation of RNA structure
„ e.g. removing cryptic splice sites and poly A signals,
optimising GC content, decreasing undesirable mRNA
folding.
slide 30
Evaluation of Gene Optimisation
Genes encoding a model antibody were gene optimised and
evaluated in stable cell lines.
„ Optimisation of the model antibody resulted in increased
mean antibody concentrations in stable cell lines.
160
Antibody
Concentration (mg/L)
„
*
140
120
100
80
60
40
20
0
41
55
nonoptimised
optimised
Mean [Ab] (mg/L)
*Significant increase
in mean [Ab] p<0.05
slide 31
Comparison of Vendor
Genes optimised by different vendors can result in different levels
of protein expression.
300
*
**
79.9
99.4
41.4
Nonoptimised
Vendor A
Vendor B
250
Antibody
concentration (mg/L)
„
200
150
100
50
0
Mean [Ab] (mg/L)
*Significant increase in mean [Ab]
**Significant decrease in mean [Ab]
slide 32
Conclusions
„
Gene optimisation can be applied to increase levels of protein
expression.
„ A solution to problems related to gene sequence.
„ Unlikely to solve issues where the protein sequence is not
optimal.
„
For manufacture of a recombinant protein, gene sequence is an
important consideration when looking to generate commercially
viable cell lines.
slide 33
Enhancing Secretion
The Mammalian SRP cycle
Nagai et al. (2003) EMBO J., 22:3479-3485
„
„
„
„
(A) SRP binds through the M domain of SRP54 to the signal sequence of membrane and secretory proteins emerging
from the exit site of the large ribosomal subunit.
(B) The Alu domain promotes transient arrest of the polypeptide chain elongation through an as yet unknown
mechanism. The affinity of SRP54 for GTP increases upon docking of SRP with the ribosome.
(C) The RNC-SRP complex diffuses to the ER membrane and docks with the SR mainly through the interaction
between SRP54 and SRα in the GTP-bound form. SRβ in the GTP-bound form interacts with the RNC complex and
induces the transfer of the signal peptide to the translocon.
(D) SRP54 and SRα mutually activate their GTPases, and SRP dissociates from the SR upon hydrolysis of GTP,
allowing the elongation of the polypeptide to resume.
slide 35
Signal Sequences
„
Antibody-derived signal sequences are often used for the
production of recombinant antibodies.
„
The amino acid composition of signal sequences can vary, but all
have similar properties (e.g. hydrophobic core).
„ It is not known how the features of a signal sequence
influence the rate protein secretion.
„
In order to evaluate if the choice of signal sequence contributes
to increased antibody concentrations, nineteen alternative
sequences were evaluated.
„ Transient expression system.
„ Stable cell lines.
slide 36
Signal Sequences
„
„
Use of vectors employing alternative sequences V1 to V19 in a transient
expression system resulted in mean antibody concentrations in the
range of 1.1 to 4.9 µg/mL.
Five candidate sequences were selected for evaluation in stable cell
lines.
Antibody
concentration (µg/mL)
6
5
4
3
2
1
0
WT WTopt V1
V2
V3
V4
V5
V6
V7
V8
V9
V10
V11 V12
V13 V14 V15 V16 V17 V18 V19
slide 37
Evaluation of signal
sequences in stable cell lines
Evaluation of the chosen sequences in stable cell lines identified
that two resulted in increased mean antibody concentrations.
73
89
100
98
105
106
300
Antibody
concentration (mg/L)
„
118
**
*
*Significant increase
in mean [Ab] p<0.05
vs WTopt
250
200
150
100
50
0
73
WT
89
100
98
105
106
118
WTopt V11
V13
V15
V16
V18
Mean [Ab] (mg/L)
slide 38
Evaluation of Product Quality
„
Are the signal sequences cleaved during the process of secretion
and not present in the final product?
„
Inappropriate processing can negatively impact on product
quality.
„ The antibody-derived signal sequences have been
previously shown to be appropriately processed in multiple
products.
„ There have been reports that incorrect processing can lead
to increased aggregation.
„
To determine if the antibody product generated using the
alternative signal sequences were appropriately cleaved purified
antibody was evaluated.
„ SDS-electrophoresis.
„ ESI-MS.
slide 39
Evaluation of Product Quality
„
Antibody generated using the alternative signal sequences was equivalent
to the material generated the antibody derived sequences (WTopt).
SDS-electrophoresis (reduced)
ESI-MS
WTopt
Light chain
Lower
markerSystem
peaks
Heavy
chain
Upper marker
Molecular Weight
(deglycosylated)
WTopt 143.5 KDa
V16
V16 143.5 KDa
V18
V18 143.5 KDa
slide 40
Conclusions
„
Choice of signal sequence can affect the level of antibody
production.
„
Equivalent product quality was observed indicating that there is
no deleterious effect on the product when using the alternative
sequences.
„
The increase observed was in addition to that seen with gene
optimisation.
„ Modulating different aspects of the protein production
pathway can result in additive benefits in recombinant protein
production.
slide 41
Comparison of Antibody Isotypes
Antibody Isotype and Expression
„
Antibodies of the same isotype can have dramatically different
expression levels.
„ The sequence of the variable region is usually selected by
the target antigen.
„ The choice of constant region is most likely to be determined
by the desired effector function for the molecule.
„
It is possible that antibody isotype should be a consideration
when attempting to maximise product yield.
slide 43
Comparison of Antibody Isotype
„
To evaluate this, GS vectors encoding a model antibody were
generated where the heavy chain was either an IgG1or IgG4
constant region.
„ Stable CHO cell lines were generated using CHOK1SV host
cells.
„ 60 cell lines were selected using Lonza’s cell line selection
strategy and evaluated in suspension shake-flask culture in a
fed-batch process designed to mimic laboratory scale
bioreactors.
slide 44
Comparison of Antibody Isotype
Mean antibody concentrations were equivalent when the
model antibody was expressed as either an IgG1 or IgG4
(p=0.178).
„ Productivity of the lead cell line for both isotypes was the
identical (2.4 g/L).
3000
Antibody
concentration (mg/L)
„
2500
2000
1500
1000
500
0
1287
1236
IgG1
IgG4
Mean [Ab] (mg/L)
slide 45
Conclusion
„
Use of an IgG1 or IgG4 isotype results in equivalent antibody
productivities.
„
„
Choice should be defined by the desired effector function for
a given product.
Variable region sequences may be the cause for the variation in
antibody productivities for antibodies of identical isotype.
slide 46
Summary
„
All aspects of the protein production pathway are important for
maximising recombinant protein expression.
„ Expression platform.
„ Gene sequence.
„ Protein sequence.
„
Choice of gene and protein sequence, along with use of leading
expression technologies and process development are crucial in
the design of commercially viable manufacturing processes.
slide 47
Acknowledgments
„
Lonza Biologics plc
„
Cell Culture Process Development
„
„
Tabitha Bullock, Rhys Davies, Salma Debar, Stephan
Kalwy, Hilary Metcalfe, Alison Porter, Helen Rogers,
Andy Racher, Rachel Sharpe, James Rance.
Analytical Development
„
Richard Aldcroft, Lisa Newey.
slide 48