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SAFC® PharmaGrade L-Methionine sulfoximine (MSX) Case Study
Confirming Cell Culture Performance for SAFC PharmaGrade MSX
In order to provide customers with a MSX product of known origin and history, SAFC has developed PharmaGrade MSX (Cat No. 76078). PharmaGrade MSX is cGMP/IPEC compliant and offers total supply chain transparency. In this study, we compare the efficacy of PharmaGrade MSX (Cat No. 76078) with the efficacy of the original Sigma-Aldrich MSX product (Cat No. M5379) by measuring inhibition of GS activity. In order to study the products’ efficacy, two methods were employed. In the first method, two producing CHO clones (that use MSX for selection pressure) were cultured in media supplemented with one of the two MSX products for 4 weeks. The clones were then placed in an assay to determine if both types of MSX were equally effective in preserving productivity after 4 weeks of passage. Growth characteristics during the passage phase of this experiment were recorded to confirm that no cell toxicity (non-specific to GS inhibition) was present with either MSX product. In the second method, a parental cell line, CHO K1, was used. If GS activity within CHO K1 is completely inhibited, the cell line will not synthesize glutamine, and therefore will not be able to divide. The response of CHO K1 to each MSX product was measured by monitoring the Viable Cell Density (VCD) and percent viability of the cells cultured in media containing either MSX product 76078 or product M5379. Materials Used Cells • Clone 27, an internal, IgG-producing cell line that relies on MSX for selection pressure • Clone 89, an internal, IgG-producing cell line that relies on MSX for selection pressure • ECACC CHO K1, a suspension adapted CHO cell line
Media and Supplements • CD Fusion (Sigma-Aldrich 14365C lot# RNBB9860) • MSX (Sigma-Aldrich M5379, lot# 061M1519V) • PharmaGrade MSX (Sigma-Aldrich 76078, lot# BCBF1273V) • Glutamine (Sigma-Aldrich G7413 lot# RNBB7697) • Water (Sigma-Aldrich W3500, lot #RNBB7890) Other • ForteBio reagents, equipment and software (Pall Life Sciences, Octet Platform) • TPP tubes (Techno Plastic Products, 91050) • Multitron (Infors) • Vicell Automated Cell Counter (Beckman Coulter 731050) Note: Before media supplementation, each MSX product was resuspended to a concentration of 100mM in water, and then sterile filtered. Excess MSX was stored at -20C. Case Specifics Method 1: Analyzing the selection pressure of each MSX product using producing clones Clone 27 and Clone 89 are internal cell lines that produce a monoclonal antibody. Both utilize a GS selection system, and rely on MSX supplementation to maintain selection pressure. If the selection pressure (i.e. the GS inhibition) is reduced, the clones will produce less monoclonal antibody and exhibit different growth characteristics. Figure 1: Average Viability of Clones at Passage Average Viability at Passage 105.0
% viable
Introduction L-Methionine sulfoximine (MSX) inhibits the activity of glutamine synthetase (GS), an enzyme essential for the production of glutamine. MSX can be used as a media supplement to aid selection and amplification processes in recombinant mammalian cell lines that use GS as a selective marker.
103.0
27_76078
101.0
27_M5379
99.0
89_76078
97.0
89_M5379
95.0 0
5
10
15
20
25
30
Day
This graph shows the average viability of each condition during the 4 week passage. There is no significant difference in either clone between cultures supplemented with MSX product 76078 or M5379. This data represents the average of the duplicates for each passage condition.
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SAFC® PharmaGrade L-Methionine sulfoximine (MSX) Case Study Figure 3: Producing Clone Viability Producing Clone Viability 100.0 27_76078B
60.0
27_M5379A 27_M5379B
40.0
89_76078A
20.0
Figure 2: Average Viable Cell Density (VCD) at Passage
89_76078B
0.0
Average VCD at Passage
89_M5379A 0
2
4
6
8
10
12
89_M5379B
Day
5.00
27_76078
4.00
27_M5379
3.00
89_76078
2.00
This graph shows the viability of each passage culture, averaged between the assay duplicates. There is no significant difference in either clone between cultures that had been supplemented with MSX product 76078 or M5379.
89_M5379
1.00 0.00 0
5
10
15
20
25
30
Day
This graph shows the average VCD of each condition during the 4 week passage. There is no significant difference in either clone between cultures supplemented with MSX product 76078 or M5379. This data represents the average of the duplicates for each passage condition.
After 4 weeks of passage, the cultures were placed in a batch growth and productivity assay, in biological duplicates (a total of 16 cultures). The assay was inoculated at a VCD of 0.3e6 cells/ ml in 25 ml of CD Fusion with no supplements, and continued for 10 days. The cultures were counted via a Vicell on days 3, 5, 7, and 10, and supernatant was harvested on days 7 and 10 to analyze IgG productivity levels. Monoclonal antibody production was measured using the Forte Bio. Supernatants from day 7 and 10 were analyzed in technical duplicates. Method 2: Analyzing the efficacy of each MSX product via inhibition of endogenous GS ECACC CHO K1 is a suspension-adapted parental cell line. Historically, supplementation of MSX at a concentration of 25µM completely inhibits GS in CHO K1, rendering the cell incapable of producing glutamine and completely inhibiting cell proliferation. To test the efficacy of the products, CHO K1 cells were placed in a growth assay. Four conditions, each in duplicate, were analyzed. In the first condition, 4mM glutamine was supplemented into CD Fusion.
In the second condition, no glutamine was supplemented, and in the third and forth conditions, 25µM of either MSX product 76078 or MSX product M5379 was supplemented. Cells were inoculated at a concentration of 0.22e6 cells/ml in 25ml of media. Viability and VCD were monitored for 10 days. Results from Method 1 Over 4 weeks of culture, no significant differences in VCDs or percent viabilities were observed between the cultures in MSX product 76078 or MSX product M5379 (see Figures 1 and 2). This indicates that the performance of both MSX products is comparable over an extended culture length; if one MSX product was more effective at inhibiting GS activity, then we would expect the clones in that condition to grow more slowly than in the less effective MSX. The similar growth characteristics also indicate that neither of the MSX products are inherently toxic to the cells (not related to GS inhibition). The results of the growth and productivity assay also confirmed that there is no difference between the two products in terms Figure 4: Producing Clone VCD Producing Clones VCD
VCD (1e6/ml)
1e6 cells/ml
27_76078A
80.0
Viability
In order to evaluate the two MSX products, each clone was cultured for 4 weeks in two different conditions: CD Fusion supplemented with 25µM MSX product 76078, and CD Fusion supplemented with 25µM MSX product M5379. Each condition was cultured in biological duplicates, for a total of 8 cultures. Each culture was maintained in TPP tubes, and passaged twice weekly to a VCD of 0.3e6 cells/ml. Cell growth and viability were tracked during this process using a Vicell.
9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00
27_76078A 27_76078B 27_M5379A 27_M5379B 89_76078A 89_76078B 89_M5379A 0
2
4
6
8
10
12
89_M5379B
Day
This graph shows the viability of each passage culture, averaged between the assay duplicates. There is no significant difference in either clone between cultures that had been supplemented with MSX product 76078 or M5379.
safcglobal.com
SAFC® PharmaGrade L-Methionine sulfoximine (MSX) Case Study of efficacy. During the batch assay, no significant difference in growth or viability was observed between cells cultured in either MSX product (Figure 3 and 4). Figure 5 shows the productivity data on days 7 and 10 of the assay. The error bars represent the standard deviation between the averaged assay titers for each of the flasks that were cultured for 4 weeks. There was no significant difference in productivity between clones cultured in each MSX product, demonstrating that both MSX product 76078 and MSX product M5379 are equally efficacious.
of the MSX products in terms of preservation of monoclonal antibody productivity in clones that use glutamine sythetase as a selective marker and MSX as a selective pressure. Method 1 also confirmed that no non-specific cell toxicity was associated with either of the MSX products. Method 2 tested the efficacy of the MSX products in terms of endogenous GS inhibition. The results of both Method 1 and Method 2 indicate that the two MSX products demonstrate equivalent efficacy in a cell culture environment. Figure 6: CHO K1 VCD Data
Figure 5: Average Assay Titers
27/M5379 89/76078
VCD (1e6/ml)
27/76078
800 600 400 200 0
89/M5379
K1 78076 K1 M5379
8.00 6.00 4.00 2.00 0.00 0
Day 7
2
4
Day 10
Results from Method 2 The results of method 2 confirm the results of method 1; there is no significant difference in efficacy between the MSX products. The control condition, CHO K1 in media supplemented with 4mM glutamine, grew as expected, with a peak cell density of 7.74e6 cells/ml (Figure 6). CHO K1 in media that did not contain glutamine reached a peak cell density of 3.35e6 cells/ml. Neither of the cultures supplemented with the MSX products displayed an increase in cell densities, and the cell viabilities of these cultures were less than observed in the glutamine supplemented and no-glutamine conditions (Figure 7). This indicates that both MSX products are effective at inhibiting endogenous GS activity. Conclusion The efficacy of PharmaGrade MSX in a cell culture environment was tested against the efficacy of a currently available MSX product. Two different methods were employed to compare the performance the materials. Method 1 tested the efficacy
8
10
12
This graph demonstrates the average VCD of CHO K1 cells over a period of 10 days, when cultured in various medias. CHO K1 survives in media containing no glutamine, showing that it contains functional GS. When either MSX product is supplemented, however, CHO K1 does not grow, indicating that both MSX products are effective at inhibiting GS activity within the cell line.
Figure 7: CHO K1 Viability Data K1 no GLN
K1 Viability
K1 GLN K1 78076
100.0
K1 M5379
90.0 80.0 70.0 60.0 50.0 0
2
4
6
8
10
12
Day
This graph demonstrates the average viability of CHO K1 over a period of 10 days, when cultured in various medias. CHO K1 survives in media containing no glutamine, showing that it contains functional GS. When either MSX product is supplemented, however, CHO K1 demonstrates a significantly lower viability, indicating that both MSX products are effective at inhibiting GS activity within the cell line.
SAFC’s PharmaGrade Products
safcglobal.com
6 Day
This graph shows the averaged titers for each original passage condition (Clone 27 in each MSX product, and Clone 89 in each MSX product). There is no significant difference in titers after the clones were cultured in media containing each MSX product.
ONS 78516-512440 1032
K1 GLN
10.00
Viability
IgG mg/L
1600 1400 1200 1000
K1 no GLN
K1 VCD Data
Average Assay Titers from each Passage Condition (clone/product number)
Contact SAFC
©2012 SAFC. All rights reserved. SAFC and SIGMA-ALDRICH are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the SAFC website at www.safcglobal.com and/or on the reverse side of the invoice or packing slip.
Confirming Cell Culture Performance for SAFC PharmaGrade MSX
In order to provide customers with a MSX product of known origin and history, SAFC has developed PharmaGrade MSX (Cat No. 76078). PharmaGrade MSX is cGMP/IPEC compliant and offers total supply chain transparency. In this study, we compare the efficacy of PharmaGrade MSX (Cat No. 76078) with the efficacy of the original Sigma-Aldrich MSX product (Cat No. M5379) by measuring inhibition of GS activity. In order to study the products’ efficacy, two methods were employed. In the first method, two producing CHO clones (that use MSX for selection pressure) were cultured in media supplemented with one of the two MSX products for 4 weeks. The clones were then placed in an assay to determine if both types of MSX were equally effective in preserving productivity after 4 weeks of passage. Growth characteristics during the passage phase of this experiment were recorded to confirm that no cell toxicity (non-specific to GS inhibition) was present with either MSX product. In the second method, a parental cell line, CHO K1, was used. If GS activity within CHO K1 is completely inhibited, the cell line will not synthesize glutamine, and therefore will not be able to divide. The response of CHO K1 to each MSX product was measured by monitoring the Viable Cell Density (VCD) and percent viability of the cells cultured in media containing either MSX product 76078 or product M5379. Materials Used Cells • Clone 27, an internal, IgG-producing cell line that relies on MSX for selection pressure • Clone 89, an internal, IgG-producing cell line that relies on MSX for selection pressure • ECACC CHO K1, a suspension adapted CHO cell line
Media and Supplements • CD Fusion (Sigma-Aldrich 14365C lot# RNBB9860) • MSX (Sigma-Aldrich M5379, lot# 061M1519V) • PharmaGrade MSX (Sigma-Aldrich 76078, lot# BCBF1273V) • Glutamine (Sigma-Aldrich G7413 lot# RNBB7697) • Water (Sigma-Aldrich W3500, lot #RNBB7890) Other • ForteBio reagents, equipment and software (Pall Life Sciences, Octet Platform) • TPP tubes (Techno Plastic Products, 91050) • Multitron (Infors) • Vicell Automated Cell Counter (Beckman Coulter 731050) Note: Before media supplementation, each MSX product was resuspended to a concentration of 100mM in water, and then sterile filtered. Excess MSX was stored at -20C. Case Specifics Method 1: Analyzing the selection pressure of each MSX product using producing clones Clone 27 and Clone 89 are internal cell lines that produce a monoclonal antibody. Both utilize a GS selection system, and rely on MSX supplementation to maintain selection pressure. If the selection pressure (i.e. the GS inhibition) is reduced, the clones will produce less monoclonal antibody and exhibit different growth characteristics. Figure 1: Average Viability of Clones at Passage Average Viability at Passage 105.0
% viable
Introduction L-Methionine sulfoximine (MSX) inhibits the activity of glutamine synthetase (GS), an enzyme essential for the production of glutamine. MSX can be used as a media supplement to aid selection and amplification processes in recombinant mammalian cell lines that use GS as a selective marker.
103.0
27_76078
101.0
27_M5379
99.0
89_76078
97.0
89_M5379
95.0 0
5
10
15
20
25
30
Day
This graph shows the average viability of each condition during the 4 week passage. There is no significant difference in either clone between cultures supplemented with MSX product 76078 or M5379. This data represents the average of the duplicates for each passage condition.
safcglobal.com
SAFC® PharmaGrade L-Methionine sulfoximine (MSX) Case Study Figure 3: Producing Clone Viability Producing Clone Viability 100.0 27_76078B
60.0
27_M5379A 27_M5379B
40.0
89_76078A
20.0
Figure 2: Average Viable Cell Density (VCD) at Passage
89_76078B
0.0
Average VCD at Passage
89_M5379A 0
2
4
6
8
10
12
89_M5379B
Day
5.00
27_76078
4.00
27_M5379
3.00
89_76078
2.00
This graph shows the viability of each passage culture, averaged between the assay duplicates. There is no significant difference in either clone between cultures that had been supplemented with MSX product 76078 or M5379.
89_M5379
1.00 0.00 0
5
10
15
20
25
30
Day
This graph shows the average VCD of each condition during the 4 week passage. There is no significant difference in either clone between cultures supplemented with MSX product 76078 or M5379. This data represents the average of the duplicates for each passage condition.
After 4 weeks of passage, the cultures were placed in a batch growth and productivity assay, in biological duplicates (a total of 16 cultures). The assay was inoculated at a VCD of 0.3e6 cells/ ml in 25 ml of CD Fusion with no supplements, and continued for 10 days. The cultures were counted via a Vicell on days 3, 5, 7, and 10, and supernatant was harvested on days 7 and 10 to analyze IgG productivity levels. Monoclonal antibody production was measured using the Forte Bio. Supernatants from day 7 and 10 were analyzed in technical duplicates. Method 2: Analyzing the efficacy of each MSX product via inhibition of endogenous GS ECACC CHO K1 is a suspension-adapted parental cell line. Historically, supplementation of MSX at a concentration of 25µM completely inhibits GS in CHO K1, rendering the cell incapable of producing glutamine and completely inhibiting cell proliferation. To test the efficacy of the products, CHO K1 cells were placed in a growth assay. Four conditions, each in duplicate, were analyzed. In the first condition, 4mM glutamine was supplemented into CD Fusion.
In the second condition, no glutamine was supplemented, and in the third and forth conditions, 25µM of either MSX product 76078 or MSX product M5379 was supplemented. Cells were inoculated at a concentration of 0.22e6 cells/ml in 25ml of media. Viability and VCD were monitored for 10 days. Results from Method 1 Over 4 weeks of culture, no significant differences in VCDs or percent viabilities were observed between the cultures in MSX product 76078 or MSX product M5379 (see Figures 1 and 2). This indicates that the performance of both MSX products is comparable over an extended culture length; if one MSX product was more effective at inhibiting GS activity, then we would expect the clones in that condition to grow more slowly than in the less effective MSX. The similar growth characteristics also indicate that neither of the MSX products are inherently toxic to the cells (not related to GS inhibition). The results of the growth and productivity assay also confirmed that there is no difference between the two products in terms Figure 4: Producing Clone VCD Producing Clones VCD
VCD (1e6/ml)
1e6 cells/ml
27_76078A
80.0
Viability
In order to evaluate the two MSX products, each clone was cultured for 4 weeks in two different conditions: CD Fusion supplemented with 25µM MSX product 76078, and CD Fusion supplemented with 25µM MSX product M5379. Each condition was cultured in biological duplicates, for a total of 8 cultures. Each culture was maintained in TPP tubes, and passaged twice weekly to a VCD of 0.3e6 cells/ml. Cell growth and viability were tracked during this process using a Vicell.
9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00
27_76078A 27_76078B 27_M5379A 27_M5379B 89_76078A 89_76078B 89_M5379A 0
2
4
6
8
10
12
89_M5379B
Day
This graph shows the viability of each passage culture, averaged between the assay duplicates. There is no significant difference in either clone between cultures that had been supplemented with MSX product 76078 or M5379.
safcglobal.com
SAFC® PharmaGrade L-Methionine sulfoximine (MSX) Case Study of efficacy. During the batch assay, no significant difference in growth or viability was observed between cells cultured in either MSX product (Figure 3 and 4). Figure 5 shows the productivity data on days 7 and 10 of the assay. The error bars represent the standard deviation between the averaged assay titers for each of the flasks that were cultured for 4 weeks. There was no significant difference in productivity between clones cultured in each MSX product, demonstrating that both MSX product 76078 and MSX product M5379 are equally efficacious.
of the MSX products in terms of preservation of monoclonal antibody productivity in clones that use glutamine sythetase as a selective marker and MSX as a selective pressure. Method 1 also confirmed that no non-specific cell toxicity was associated with either of the MSX products. Method 2 tested the efficacy of the MSX products in terms of endogenous GS inhibition. The results of both Method 1 and Method 2 indicate that the two MSX products demonstrate equivalent efficacy in a cell culture environment. Figure 6: CHO K1 VCD Data
Figure 5: Average Assay Titers
27/M5379 89/76078
VCD (1e6/ml)
27/76078
800 600 400 200 0
89/M5379
K1 78076 K1 M5379
8.00 6.00 4.00 2.00 0.00 0
Day 7
2
4
Day 10
Results from Method 2 The results of method 2 confirm the results of method 1; there is no significant difference in efficacy between the MSX products. The control condition, CHO K1 in media supplemented with 4mM glutamine, grew as expected, with a peak cell density of 7.74e6 cells/ml (Figure 6). CHO K1 in media that did not contain glutamine reached a peak cell density of 3.35e6 cells/ml. Neither of the cultures supplemented with the MSX products displayed an increase in cell densities, and the cell viabilities of these cultures were less than observed in the glutamine supplemented and no-glutamine conditions (Figure 7). This indicates that both MSX products are effective at inhibiting endogenous GS activity. Conclusion The efficacy of PharmaGrade MSX in a cell culture environment was tested against the efficacy of a currently available MSX product. Two different methods were employed to compare the performance the materials. Method 1 tested the efficacy
8
10
12
This graph demonstrates the average VCD of CHO K1 cells over a period of 10 days, when cultured in various medias. CHO K1 survives in media containing no glutamine, showing that it contains functional GS. When either MSX product is supplemented, however, CHO K1 does not grow, indicating that both MSX products are effective at inhibiting GS activity within the cell line.
Figure 7: CHO K1 Viability Data K1 no GLN
K1 Viability
K1 GLN K1 78076
100.0
K1 M5379
90.0 80.0 70.0 60.0 50.0 0
2
4
6
8
10
12
Day
This graph demonstrates the average viability of CHO K1 over a period of 10 days, when cultured in various medias. CHO K1 survives in media containing no glutamine, showing that it contains functional GS. When either MSX product is supplemented, however, CHO K1 demonstrates a significantly lower viability, indicating that both MSX products are effective at inhibiting GS activity within the cell line.
SAFC’s PharmaGrade Products
safcglobal.com
6 Day
This graph shows the averaged titers for each original passage condition (Clone 27 in each MSX product, and Clone 89 in each MSX product). There is no significant difference in titers after the clones were cultured in media containing each MSX product.
ONS 78516-512440 1032
K1 GLN
10.00
Viability
IgG mg/L
1600 1400 1200 1000
K1 no GLN
K1 VCD Data
Average Assay Titers from each Passage Condition (clone/product number)
Contact SAFC
©2012 SAFC. All rights reserved. SAFC and SIGMA-ALDRICH are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the SAFC website at www.safcglobal.com and/or on the reverse side of the invoice or packing slip.
