Diploid SFM Update and VOC

Anna Hachmann, Abhijeet Kohli

For Research Use or Further Manufacturing. Not for diagnostic use or direct administration into humans or animals. The world leader in serving science
Update on Diploid SFM and Application Data

• We developed Diploid SFM using metabolite analysis and a design of experiment rationale for growth of
MRC-5 and other fibroblast cells. It can support adaptation-free expansion, while resulting in performance
that is comparable to serum-containing medium.

• Since requirements for production of viruses are different from cell growth, we optimized the production
medium separately. This animal origin-free production medium is designed to allow manufacturers to
produce vaccines without concern about the bovine serum albumin limit of 50 ng/dose set by the World
Health Organization.

• Diploid SFM was shown to support growth of diploid and other fibroblast cell lines: MRC-5, WI-38, CEF,
KMB-17, SV-1 and production of VZV, VSV, coxsackievirus, and cytomegalovirus.

• Some processes may benefit from supplementation with 1% FBS during growth, while maintaining an
animal origin-free virus production process (see data in following slides).

• We confirmed virus production with VZV and vesicular stomatitis virus and demonstrate titers that are
comparable to a classical medium control. By switching to a serum-free or low-serum process, vaccine
manufacturers can reduce dependency on serum, production and purification costs, and increase product
consistency and safety.

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 MRC-5 Viable Cell Density and VSV Production at Higher Seeding Density

MRC-5 Viable Cell Density VSV Titer

Seeding Density 1.5x104 cells/cm2 - 4-day Passage Seeding Density 1.5x104 cells/cm2
8.E+04 1.E+08

7.E+04 1.E+07

6.E+04 1.E+06
Cells/cm2

5.E+04 1.E+05

TCID50/mL
4.E+04 1.E+04

3.E+04 1.E+03

2.E+04 1.E+02

1.E+04 1.E+01

0.E+00 1.E+00
MEM Alpha DSFM DSFM DSFM MEM Alpha DSFM DSFM DSFM
+ 10% FBS + 1% FBS + 50% Spent Medium + 10% FBS + 1% FBS + 50% Spent Medium

• Evaluation of MRC-5 viable cell density and VSV production at a seeding density of 1.5x104 cells/cm2. Cells were passaged
every 4 days for 5 passages at the indicated conditions before infection with VSV at MOI 0.01. The medium was exchanged
to Diploid Production Medium (or classical medium control) for infection. Virus was harvested 2 days post infection.
• Diploid SFM supplementation with 1% FBS or 50% spent medium (serum-free) during growth increased VSV production.

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 MRC-5 Viable Cell Density and VSV Production at Lower Seeding Density

MRC-5 Viable Cell Density VSV Titer

Seeding Density 0.8x104 cells/cm2 - 4-day Passage Seeding Density 0.8x104 cells/cm2
6.E+04 1.E+08

1.E+07
5.E+04
1.E+06
4.E+04
Cells/cm2

1.E+05

TCID50/mL
3.E+04 1.E+04

1.E+03
2.E+04
1.E+02
1.E+04
1.E+01

0.E+00 1.E+00
MEM Alpha DSFM DSFM DSFM MEM Alpha DSFM DSFM DSFM
+ 10% FBS + 1% FBS + 50% spent medium + 10% FBS + 1% FBS + 50% spent medium

• Evaluation of MRC-5 viable cell density and VSV production at a seeding density of 0.8x104 cells/cm2. Cells were passaged
every 4 days for 5 passages at the indicated conditions before infection with VSV at MOI 0.01. The medium was exchanged
to Diploid Production Medium (or classical medium control) for infection. Virus was harvested 2 days post infection.
• Diploid SFM supplementation with 1% FBS or 50% spent medium (serum-free) during growth increased VSV production.

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 MRC-5 Seed Train Simulation over 4 Passages and VSV Production

MRC-5 Seed Train Simulation Total VSV Per Production Process

Seeding Density 1.5x104 cells/cm2 Seeding Density 1.5x104 cells/cm2
160 1.E+11

MEM Alpha + 10% FBS 1.E+10

140
DSFM 1.E+09

PFU/Production Process
120
1.E+08
DSFM + 1% FBS
Total # of Vessels

100 1.E+07
DSFM + 50% spent medium
1.E+06
80
1.E+05
60 1.E+04
1.E+03
40
1.E+02
20
1.E+01

0 1.E+00
1 2 3 4 MEM Alpha DSFM DSFM DSFM
Passage # + 10% FBS + 1% FBS + 50% spent medium

Evaluation of MRC-5 scale-up process simulation:

• Seeding density 1.5x104 cells/cm2 in T-225 flasks, 4-day passages, 4-passage scale-up, VSV infection at MOI 0.01
• Starting with 1 T-225 flask at Passage 1, up to 127 T-225 flasks could be generated in Diploid SFM + 1% FBS over 4
passages, and up to 7.41x1010 VSV PFU could be produced with Diploid Production Medium.

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 MRC-5 Seed Train Simulation over 4 Passages and VSV Production

MRC-5 Seed Train Simulation Total VSV Per Production Process

Seeding Density 0.8x104 cells/cm2 Seeding Density 0.8x104 cells/cm2
250 1.E+12
MEM Alpha + 10% FBS 1.E+11

200 DSFM 1.E+10

1.E+09

PFU/Production Process
DSFM + 1% FBS
Total # of Vessels

1.E+08
150 DSFM + 50% spent medium
1.E+07
1.E+06
100 1.E+05
1.E+04
1.E+03
50
1.E+02
1.E+01
0 1.E+00
1 2 3 4 MEM Alpha DSFM DSFM DSFM
Passage # + 10% FBS + 1% FBS + 50% spent medium

Evaluation of MRC-5 scale-up process simulation:

• Seeding density 0.8x104 cells/cm2 in T-225 flasks, 4-day passages, 4-passage scale-up, VSV infection at MOI 0.01
• Starting with 1 T-225 flask at Passage 1, up to 192 T-225 flasks could be generated in Diploid SFM + 1% FBS over 4
passages, and up to 1.82x1011 VSV PFU could be produced with Diploid Production Medium.

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Growth Performance of Chicken Embryo Fibroblasts

Growth performance of Chicken Embryo Fibroblasts (CEF):

• Chicken Embryo Fibroblasts were extracted with trypsin, washed with DPBS with Ca2+ and Mg2+ and seeded with
Diploid SFM or MEM-H + 10% FBS at 1.5x105 cells/cm2 seeding density. Cells were incubated for 4 days.
• Diploid SFM supports CEF culture without supplementation of FBS.

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Conclusion
• Growth performance with MRC-5 in Diploid SFM compared to classical medium with serum is slightly reduced
at seeding densities of 15,000 cells/cm2 or higher. Supplementation with 1% serum or spent Diploid medium
can support growth at higher densities.

• Independent of the seeding density, the virus production with MRC-5 in the animal origin-free Diploid
production medium is comparable or higher than the control in classical medium.

• In conclusion, Diploid SFM supports growth of diploid and other fibroblast cell lines (MRC-5, WI-38, CEF, KMB-
17, SV-1). Growth with MRC-5 cells at higher seeding densities may require supplementation of 1% FBS or
spent medium. The specific virus productivity is higher in Diploid production medium compared to classical
medium control (VZV, VSV, coxsackievirus, cytomegalovirus).

• cGMP lots (ISO 13485 and 21 CFR 820) of Diploid SFM are available for customer testing

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Diploid SFM – VOC Questions
1. What cell line and virus are you producing?

2. We would like to ask you some specific questions about your process:

a) Please help us understand your seed train (scale-up process): Seeding densities, harvest densities, number of passages
and days of culture per passage?
b) What brand production vessel do you use? Size (cm2)? (Recommend Corning surface)
c) How much virus can you harvest from one production vessel? (Titer/cm2 or Titer/vessel)
d) How many days post seeding do you infect with virus?
e) How many days pass between infection and final harvest? How many medium exchanges are performed during this time?

3. Would you be able to modify your process for optimal performance with Diploid SFM?

a) Would you be able to change the seeding density (for MRC-5: 6000 – 8000 cells/cm2)?
b) Would you be able to accept a lower growth performance, as long as the virus titer remains the same?
c) Would you be able to accept supplementation with 1% FBS, as long as that provides comparable growth performance and
improved virus titer?

4. Are you interested in optimization with a microcarrier-based bioreactor process? What microcarriers are you using?

5. Are you interested in a medium for chicken embryo fibroblasts?

6. Are you interested in continuing to test with Diploid SFM given the information we have provided?

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Supplemental Slide
Population Doublings of MRC-5 at High Density
Culture
50

48

46

Cumulative Population Doublings

44

42

40

38

36 MEM Alpha + 10% FBS

34 Diploid SFM
Diploid SFM + 1% FBS
32
Diploid SFM + 50% Spent Medium
30
10 15 20 25 30 35 40 45
Days

Cumulative Population Doublings of MRC-5:

• Seeding density 1.5x104 cells/cm2, 3 x 3-day passages followed by 4 x 7-day passages in the indicated media
• Diploid SFM + 1% FBS performed comparable to or better than the control (MEM Alpha + 10% FBS)

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