designing generic high-yielding processes for gs...
TRANSCRIPT
INTRODUCTION
Mammalian cell lines are widely used by the pharmaceutical industry for the production of therapeutic proteins. The identification of a high producing recombinant mammalian line is a key economic consideration.
Over the past few years, we have developed a series of processes for cell lines using the Glutamine Synthetase Gene Expression System (Lonza Biologics) to allow the rapid generation of material for the clinic. These processes, performed in chemically defined and animal component-free (CDACF) medium, were improved to meet ever increasing market demands for high yielding processes. We investigated parameters affecting the growth and productivity of Chinese Hamster Ovary (CHO) cells.
Two routes were chosen to improving process productivity:
• Increase the time integral of viable cell concentration (IVC)
• Increase the specific antibody production rate of the cells (Qp)
Furthermore, product quality was profiled during the course of a fermentation using the optimised process to verify that product quality was not compromised through extension of the culture duration. These data were also compared with product quality data from the original process.
MATERIALS AND METHODSCell Lines• LB01 generated by transfecting host cell line CHOK1SV with GS vector encoding the
genes for IgG4 antibody heavy chain and light chain, adapted to growth in CDACF media.
Media and Feeds• GS-CHO: CD-CHO from Invitrogen.• Proprietary CDACF feeds.
Bioreactor Processes• 10 L airlift bioreactors operated with on-line control of dissolved oxygen, temperature and
pH.• Product concentration was determined by Protein A HPLC.• Product quality was profiled by SDS-PAGE, IEF, MALDI-TOF and tryptic peptide mapping.
RESULTS AND DISCUSSIONProcess Optimisation using pH DriftA model GS-CHO cell line (LB01) was grown at a pH below pH 7.0. Reducing the culture pH increased the culture duration (Figure 1) and IVC (Figure 2). Additionally the Qp was increased throughout the culture resulting in an increased harvest antibody concentration of 4.3 g/L (Figure 2). A reduction in lactate production with a pH drift was also apparent in this optimised process, suggesting a shift in cell metabolism (Figure 3). This reduction in lactate accumulation also provided a further benefit through a significantly decreased need for alkali addition.
Designing Generic High-Yielding Processes for GS-CHO Cell LinesM. H. Rendall, A. Maxwell, A. Pham, M.J. Davies and D. O. Mainwaring
Lonza Biologics plc, 228 Bath Road, Slough, Berkshire SL1 4DX, UK Figure 1. Profile of cell growth in optimised versus original process for the
GS-CHO cell line LB01.
Figure 2. Profile of the productivity of the optimised versus original using the GS-CHO cell line LB01.
Figure 3. Profile of glucose and lactate of the optimised versus original process using the GS-CHO cell line LB01.
Product Quality Profiling
In parallel with improvements in growth and productivity of the GS-CHO process, product quality consistency was profiled throughout the extended culture duration of the optimised process. The biochemical characteristics of this antibody were compared at different stages throughout the fermentation process. Biochemical comparability was assessed by SDS-PAGE (Figure 4), IEF (Figure 5), MALDI TOF-MS (Figure 6) and RP HPLC tryptic peptide mapping (Figure 7) of samples purified by Protein A affinity chromatography.
Original Process Optimised Process
Elapsed Time (h)
Lact
ate
Conce
ntr
ation (
g/L
)
0
1
2
3
4
5
6
7
8
0 48 96 144 192 240 288 336 384 432 480 528
Figure 4. Affect of culture duration on molecular weight of IgG4 antibody as determined by non-reduced SDS-PAGE of the optimised process. Original process profile also shown for comparison.
Banding patterns (Figure 4) observed were consistent throughout the fermentation (day 7 to day 20). A slight increase in the overall proportion of whole antibody was observed over the course of the fermentation, and was associated with a decrease in the levels of half antibody. It is currently unknown why the proportion of half antibody was reduced, or furthermore whether the change was pre- or post-secretion.
Figure 5. IEF profiles for an IgG4 antibody produced by the GS-CHO cell line LB01 between day 9 and day 20 of the fermentation process. The original process IEF profile is also shown for comparison.
IEF banding profiles were consistent throughout the fermentation to day 20. Furthermore these profiles are consistent with the original process.
Figure 6. MALDI-TOF Oligosaccharide profiles for an IgG4 antibody produced from the GS-CHO cell line LB01, between day 7 and day 20 of the optimised fermentation process.
The antibody glycosylation profiles determined by MALDI-TOF mass spectrometry were predominantly bi-antennary core fucosylated glycans with varying amounts of terminal galactose (G2F, G1F and G0F). A slight decrease in the degree of terminal galactosylation was observed up to day 13, however, this decrease did not exceed the variation observed as part of typical batch to batch variability. The oligosaccharide profiles observed were also comparable to those for the original process (data not shown). It is under investigation why terminal galactose decreased during the fermentation, possible causes included reduced availability of substrate or variation in galactosyltransferase activity. Further manipulation of nutrient feed regimes or the physiochemical environment may be required to modify late culture galactosylation profiles.
Figure 7. RP HPLC tryptic peptide map profiles for an IgG4 produced in the GS-CHO cell line LB01 from day 9 to day 20 of the optimised fermentation.
pI
Mark
ers
Day
9
Day
11
Day
13
Day
15
Day
16
Day
17
Day
18
Day
19
Day
20
Origin
al
pro
ces s
Bla
nkpI
Mark
er s pI
Mark
ers pI
Mark
er s
1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150m/z0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
AP01040423 7 (0.594) Cm (1:9) TOF LD+ 6.96e31485.69
1339.68
1647.78
1527.79 1809.88
AP01040422 4 (0.319) Cm (1:9) TOF LD+ 4.19e31485.67
1339.601123.82
1647.52
1527.73 1809.82
AP01040421 7 (0.599) Cm (1:9) TOF LD+ 2.56e31485.72
1127.10 1283.091201.01 1339.65
1647.80
1527.81 1809.941689.82
AP01040420 5 (0.348) Cm (1:9) TOF LD+ 9.13e31485.67
1339.58
1647.72
1527.71 1809.82
AP01040419 4 (0.317) Cm (1:9) TOF LD+ 9.56e31485.61
1339.58
1647.66
1527.62 1809.761689.68
Day 16
Day 17
Day 18
Day 19
Day 20
G0F G1FG2F
G0F G1FG2F
G1F G2F
G0F G1FG2F
G0F G1FG2F
G0F
1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150m/z0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
AP01040418 1 (0.041) Cm (1:9) TOF LD+ 6.97e31485.64
1339.55
1647.69
1527.62 1809.761689.77
AP01040417 6 (1.268) Cm (1:9) TOF LD+ 2.08e31485.64
1139.30 1339.551281.34
1647.72
1527.62 1809.731689.79
AP01040416 1 (0.041) Cm (1:9) TOF LD+ 5.14e31485.61
1340.541178.51 1384.59
1647.69
1527.651809.76
1689.71
AP01040415 7 (1.127) Cm (1:9) TOF LD+ 2.88e31485.64
1129.861173.871217.96 1339.601282.57
1647.69
1527.681809.76
1689.77
AP01040414 3 (0.804) Cm (1:9) TOF LD+ 3.71e31647.69
1485.64
1340.561129.63 1282.55 1527.57
1809.76
1689.74
G0F
G0F
G0F
G0F
G0F
G1F
G1F
G1F
G1F
G1F
G2F
G2F
G2F
G2F
G2F
Day 15
Day 13
Day 11
Day 9
Day 7
1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150m/z0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
AP01040423 7 (0.594) Cm (1:9) TOF LD+ 6.96e31485.69
1339.68
1647.78
1527.79 1809.88
AP01040422 4 (0.319) Cm (1:9) TOF LD+ 4.19e31485.67
1339.601123.82
1647.52
1527.73 1809.82
AP01040421 7 (0.599) Cm (1:9) TOF LD+ 2.56e31485.72
1127.10 1283.091201.01 1339.65
1647.80
1527.81 1809.941689.82
AP01040420 5 (0.348) Cm (1:9) TOF LD+ 9.13e31485.67
1339.58
1647.72
1527.71 1809.82
AP01040419 4 (0.317) Cm (1:9) TOF LD+ 9.56e31485.61
1339.58
1647.66
1527.62 1809.761689.68
Day 16
Day 17
Day 18
Day 19
Day 20
G0F G1FG2F
G0F G1FG2F
G1F G2F
G0F G1FG2F
G0F G1FG2F
G0F
1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150m/z0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
AP01040418 1 (0.041) Cm (1:9) TOF LD+ 6.97e31485.64
1339.55
1647.69
1527.62 1809.761689.77
AP01040417 6 (1.268) Cm (1:9) TOF LD+ 2.08e31485.64
1139.30 1339.551281.34
1647.72
1527.62 1809.731689.79
AP01040416 1 (0.041) Cm (1:9) TOF LD+ 5.14e31485.61
1340.541178.51 1384.59
1647.69
1527.651809.76
1689.71
AP01040415 7 (1.127) Cm (1:9) TOF LD+ 2.88e31485.64
1129.861173.871217.96 1339.601282.57
1647.69
1527.681809.76
1689.77
AP01040414 3 (0.804) Cm (1:9) TOF LD+ 3.71e31647.69
1485.64
1340.561129.63 1282.55 1527.57
1809.76
1689.74
G0F
G0F
G0F
G0F
G0F
G1F
G1F
G1F
G1F
G1F
G2F
G2F
G2F
G2F
G2F
Day 15
Day 13
Day 11
Day 9
Day 7
Day
16
Day
17
Day
18
Day
19
Day
20
MW
Mark
ers
Day
7
Day
9
Day
11
Day
13
Day
15
MW
Mark
ers
Inte
rass
ay
Contr
ol
Origin
al
pro
cess
MW
Mark
ers
Inte
rass
ay
Contr
ol
MW
Mark
ers
MW
Mark
ers
RP HPLC tryptic peptide maps were comparable throughout the fermentation, with no new peaks being detected between days and only minor variations in peak intensity being observed. These peptide maps were also comparable to the original process (data not shown).
CONCLUSIONS• Increased productivity from mammalian cell cultures requires a combination of highly
productive cell lines and fermentation processes that improve both IVC and Qp. Reduction of the culture pH below pH 7.0 was effective as an optimisation strategy since it improved both parameters.
• In parallel with process improvement programmes, it is paramount to verify that process changes do not adversely impact product quality.- SDS PAGE and IEF profiles were comparable throughout the fermentation and consistent
with the typical profiles for an IgG. - Oligosaccharide profiling of N-glycans indicated a slight decrease in terminal galactose
during the early stage of fermentation, however this did not exceed typical acceptable product specification limits and the profiles were comparable across the fermentation and to the original process.
- RP HPLC tryptic peptide maps were comparable throughout the fermentation, with no new peaks being detected between days and only minor variations in peak intensity being observed.
• In summary, extension of the culture duration in the optimised process did not demonstrate an adverse effect on product quality.
• Overall product quality of the optimised process was comparable with the original process.
ACKNOWLEDGEMENTSCell culture process development, Lonza Biologics.Assay development, Lonza Biologics.
m0 20 40 60 80 100
mAU
0
100
200
300
400
DAD1 A, Sig= 210, 4 Ref= 360, 100 (LM260204\008-0801.D)
Day 17
m0 20 40 60 80 100
mAU
0
100
200
300
400
DAD1 A, Sig= 210, 4 Ref= 360, 100 (LM260204\009-0901.D)
Day 18
m0 20 40 60 80 100
mAU
0
100
200
300
400
DAD1 A, Sig= 210, 4 Ref= 360, 100 (LM260204\010-1001.D)
Day 19
m0 20 40 60 80 100
mAU
0
100
200
300
400
DAD1 A, Sig= 210, 4 Ref= 360, 100 (LM260204\011-1101.D)
Day 20
0 20 40 60 80 100 120m0
DAD1 A, Sig=210,4 Ref=360,100 (LM260204\003-0301.D)
Day 9
0 20 40 60 80 100 120m0
200
DAD1 A, Sig=210,4 Ref=360,100 (LM260204\004-0401.D)
Day 11
0 20 40 60 80 100 120m0
200
DAD1 A, Sig=210,4 Ref=360,100 (LM260204\005-0501.D)
Day 13
0 20 40 60 80 100 120m0
200
DAD1 A, Sig=210,4 Ref=360,100 (LM260204\006-0601.D)
Day 15
0 20 40 60 80 100 120m
mAU
0
200
DAD1 A, Sig=210,4 Ref=360,100 (LM260204\007-0701.D)Day 16
200
mAU
mAU
mAU
mAU
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
0 500 1000 1500 2000 2500 3000 3500 4000
Time Integral of Viable Cell Concentration (109 cell h/L)
Pro
duct
Conce
ntr
ation (
mg/L
)
Original Process Optimised Process
Elapsed Time (h)
Via
ble
Cel
l Conce
ntr
atio
n (
10
6 /m
L)
Original Process Optimised Process
0.1
1.0
10.0
100.0
0 48 96 144 192 240 288 336 384 432 480 528