insect media evaluation for cell growth and … · figure 2: media evaluation -product a...

75 Sidney St. Cambridge, MA 02139 USA - [email protected] - 857-259-5340

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Time (Days Post Infection)Media C (Flask) Media F (Flask)

Media G (Flask) Media C (2L STR, N=2)

Media D (2L STR, N=2) Media G (2L STR, N=3)

INSECT MEDIA EVALUATION FOR CELL GROWTH AND rAAV PRODUCTION IN AN SF9-BACULOVIRUS SYSTEMTaylor Polhemus1, Hui Wu1, David Dismuke2, and Christopher J. Morrison1

ABSTRACTExpanding clinical efforts utilizing rAAV vectors have required continuousimprovements in rAAV manufacturing. Particularly, increased productsupply demands have fueled efforts to maximize volumetric productivity ofthe cell culture process. One avenue to improve volumetric productivity isthe optimization of cell culture media. In this work we compare theperformance of a range of commercially available Sf9-specific cell culturemedia. Cells were maintained for approximately 100 population doublingsto evaluate media robustness and ensure full adaptation to the assortedmedia. Multiple rounds of vector production were performed using variousAAV serotypes to investigate the impact of the different media on thequantity and quality of vector produced. Furthermore, terminal batchgrowth cultures were conducted to investigate differences in growth andmetabolic profiles. The importance of media selection and optimization inplatform development is demonstrated in this work by marked volumetricand cellular productivity improvement for our process. These resultshighlight the significant impact that the cell culture media has on theproduction of rAAV in the Sf9-Baculovirus manufacturing system.

1Voyager Therapeutics, 75 Sidney St, Cambridge, MA 02139

Figure 1: Maintenance and Terminal Growth Curve Comparison

Overview

A

A) Flask maintenance data. Generations aredefined as a population doubling in culture.Certain conditions were terminated beforethe four production rounds were completeddue to poor growth or productivity. Media B,C, F and G were maintained for the fullduration of the experiment.

B) Subset of the terminal batch growth datafor the different media evaluated. Allconditions were seeded at 1.0E6 viablecells/mL in shake flasks. No furthersupplementation was provided over thecourse of the batch. Cell growth parameterswere generated by offline sampling usingthe Nexelcom Cellometer Auto T4(Cellometer).

Media ID Serum-Free Animal Origin Free Hydrolysate/

Yeastolates

A ✓ × ✓B ✓ ✓ ✓C ✓ ✓ ✓D ✓ ✓ ✓E ✓ × ✓F ✓ ✓ ✓G ✓ ✓ ✓

• Host Cell Line – Spodoptera frugiperda 9 (Sf9 – Insect)• Baculovirus – Expression vector to produce recombinant Adeno-Associated Virus• Product A = RH10 Capsid• Product B = AAV2 Capsid

Production of rAAV in the Sf9/Baculovirus System

Evaluation: Media was evaluated over the course of 5 months for a defined set of criteria

Passaging Terminal Batch

Media ID

Total Gen.

Avg. Doubling Time (hrs.)

CV% Avg. Peak VCD(E6 cells/mL)

Length to 70% Viability

(days)

A 45.7 29.3 7.2 8.7 10

B 114 36.8 13.3 16.3 10

C 138.3 28.7 5.4 16.5 13

D 39.6 58.0 38.6 8.4 10

E 71.9 39.1 8.0 10.7 10

F 96.4 36.6 4.1 16.8 10

G 127.3 28.9 4.9 13.0 8

Figure 2: Media Evaluation - Product A • Differences in cell growth were observed

and were not directly correlated to the differences in the terminal batch growth evaluation.

• Media F was the highest producing media tested with an average qPCR titer of 1.06E12 vg/mL.

• With the exception of Media E, cell line age (Generation) could not be correlated to productivity.

• Rank order was highly conserved whether comparing productivity by volumetric or cell specific productivity.

• Volumetric Productivity –(F > E > G > B > D > C> A) vs. Cell Specific Productivity –(F > E > G > B > C > D > A)

A) Cell parameter data for the production of Product A. All conditions are representative of the platform process used toproduce Product A. The production flasks were operated at a working volume of 200 mL in a 500mL shake flask. Cultureswere infected with baculovirus on Day 0 of culture. Cell growth parameters were generated by offline sampling using theNexelcom Cellometer Auto T4 (Cellometer).

B) Volumetric Titer’s are quantified by qPCR on clarified lysate samples.

C) Cell specific productivity is defined as:!"#$%&%'( )*+#,' -%,'$ (/012)

4'#5 6%#7"' !'"" 8'9+%,* (:;<=>>?12 )

A

B C

Figure 3: Media Evaluation - Product B

CONCLUSIONS• The data presented highlight the range of performance in commercially

available Sf9 cell culture media.

• Screening commercially available cell culture media was shown toproduce a full log of dynamic range in volumetric and cell specificproductivity for two different therapeutics.

• Rank order of commercially available cell culture media was conservedacross products and scale down models (Flask vs. 2L STR).

The authors would like to acknowledge and thank the following parties for their contribution(s) to the data presented:Process Analytics/Analytical Development Group at Voyager; Process Development Group at Voyager

ACKNOWLEDGMENTS

• A range of peak viable cell densities was observed across the media tested. Peak viable cell density ranged from 6-10E6 viable cells/mL.

• Media C and G experienced an accelerated decline in viability compared to Media B and F.

• Media F consistently exhibits an inflated average cell diameter across the duration of culture compared to the other media tested.

• Ranking of media productivity by both volumetric and cell specific productivity was not impacted by the product used. Rank order was conserved across the two products tested (Media F > G > B > C).

A) Cell parameter data for the production of Product B. All conditions are representative of the platform process used toproduce Product B. The production flasks were operated at a working volume of 200 mL in a 500mL shake flask. Cultureswere infected with baculovirus on Day 0 of culture. Cell growth parameters were generated by offline sampling using theNexelcom Cellometer Auto T4 (Cellometer) .


Cell specific productivity is defined as:!"#$%&%'( )*+#,' -%,'$ (/012)

4'#5 6%#7"' !'"" 8'9+%,* (:;<=>>?12 )

A B

Figure 4: Impact of Scale on Media Evaluation

• Cell growth parameters trend consistently between flask and 2L STR for Media C and G.

• A delay in infection kinetics was observed for Media F when comparing the flask trend to the 2L STR. Peak viable cell density, viability decline, and timing of average diameter increase are delayed ~ 24 hours respectively.

• Media F was again the highest producing media on a volumetric and cell specific basis.

• Ranking of media by volumetric and cell specific productivity was not impacted by the scale used for evaluation. Rank order was conserved from the shake flask evaluation (Media F > G > C).

A) Cell parameter data for the production of Product B at the Flask (200mL Working Volume in 500 mL Flask), and 2L STRscales. All conditions are representative of the platform process used to produce Product B. The 2L STR trends areaveraged values with the number of replicates specified in the legend. Cultures were infected with baculovirus on Day 0 ofculture. Cell growth parameters were generated by offline sampling using the Nexelcom Cellometer Auto T4 (Cellometer).


C) Cell specific productivity is defined as:!"#$%&%'( )*+#,' -%,'$ (/012)

4'#5 6%#7"' !'"" 8'9+%,* (:;<=>>?12 )

A

B C

Volumetric Titer Cell Specific Titer

Cell Parameters

Titer CombinedCell Parameters


Cell Parameters

Purified rAAV

Various Purification Operations

Infection with baculovirus

Production occurs in various vessels

Sf9 cells cultured in suspension

Purification of harvest

• The average doubling times of Sf9 cells in Media A, C, and G fell within the average literature range of 24-30 hours. The average doubling times of Sf9 cells in Media B, D, E and F exceeded the average literature range.

• Media F and G exhibited the most consistent doubling times with a CV of less than 5% over the maintenance timeframe.

• Media C displayed the most robust performance in the terminal batch growth evaluation. The media was able to support a high maximum viable cell density (avg. 16.5E6 viable cells/mL) while maintaining high viability (> 80%) through Day 13 in terminal batch growth.

• Adaptation and StabilityCells were passaged in respective media for ~100 generations• Terminal Batch Growth

Compare peak densities in batch growth• Production Comparison

Four rounds of rAAV Production- Compare productivity and quality attributes

B

Media Summary

Passaging Summary

Cell Parameters

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Experimental Range = ~1E11 - 1E12 vg/mL

Experimental Range = ~1E4 - 2E5 vg/cell

NOTES: 2Formerly of Voyager Therapeutics, currently at StrideBio, Durham, NC

Abstract #640ASGCT 2018

insect media evaluation for cell growth and … · figure 2: media evaluation -product a...

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