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RoosterReplenishTM-MSC-XFR

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ABSTRACT

• With a growing demand for hMSCs to support late phase clinical trials, the approach

to manufacture hMSCs on a large scale under current Good Manufacturing Practice

(cGMP) regulation is highly important.

• Current large scale hMSC manufacturing involves monolayer cell expansion in multi-

layer vessels; however, scalability of this process is challenging. Microcarrier

suspension bioreactor systems provide an alternative method to scale up hMSC

production to meet the required lot sizes necessary to support clinical trials.

• The Quality by Design (QbD) approach was utilized to develop a scalable xeno-free

(XF) hMSC bioreactor process that maintains the final cell population doubling level

(PDL) within the recommended range (16-20) to ensure product quality, even as the

bioreactor culture is scaled.

• The scalability of culturing XF hBM-MSCs in low sheer single-use, Vertical-Wheel

suspension bioreactors (PBS Biotech) was evaluated at the small (0.1L), development

(3L), pilot (15L) and production (50L) scale.

• These development studies show that XF hMSCs can effectively be expanded in a

scalable bioreactor culture system, which allows for significant time and cost savings

in a standardized system for use in the regenerative medicine, tissue engineering,

and cell therapy fields.

SCALING A XENO-FREE (XF) FED-BATCH MICROCARRIER SUSPENSION BIOREACTOR SYSTEM TO THE PRODUCTION SCALE (50L) FOR

MANUFACTURING XF hMSCsRobert Kirian1, David Wang1, Josephine Lembong1, Joseph Takacs1, Michelle Trempel1, Ang-Chen Tsai2, Kenny Cruz2, Francisco Rosello2, Kayley Cox2, Yas Hashimura2,

Jon Rowley1, Sunghoon Jung2, Taby Ahsan1

1 RoosterBio, Inc. 5295 Westview Drive, Suite 275, Frederick, MD 217032 PBS Biotech, Inc. 1183 Calle Suerte, Camarillo, CA 93012

COMPARABLE CELL & MICROCARRIER AGGREGATION

SUMMARY

• This study has shown that a fed-batch bioreactor process enhances mediaproductivity, is more cost-effective, and less labor-intensive for large scale expansionof hMSCs in suspension culture.

• hMSC yields of up to 500,000 cells/mL were achieved within 4 - 5 days at the small(0.1L), development(3L), pilot(15L), and production (50L) bioreactor scales utilizing afed batch xeno-free culture process.

• hMSC expanded and harvested in the 50L bioreactor maintained similar phenotypicand functional properties compared to monolayer cells cultured in parallel.

• The comparable cell seeding distribution, growth curve, biochemical profiles duringculture, and final harvest yields demonstrate a scalable bioreactor process (0.1L – 50L)that may be further scaled to larger bioreactor scales that are necessary to generatethe cell yields for clinical therapies.

5295 Westview Drive, Suite 275, Frederick, MD 21703 USA

Seed hMSCs on Microcarriers

Add Feed toCulture

Harvest and Vial hMSCs

Day 0 Day 3 Day 5

EVOLUTION OF hMSC MANUFACTURING PLATFORM

• hMSC manufacturing platforms need to evolve as the demand for cells to supportlate phase clinical trials increases.

• Producing cells in T-flasks and multi-layer vessels are inefficient at lot sizes of >1B and>30B cells, respectively, as the amount of time and labor required during culture andharvest becomes impractical.

• Suspension bioreactors are one type of manufacturing platform that can provide thenecessary lot sizes of hundreds-of-billions to trillions of cells while reducing the time,labor, and cost of goods (COGs) for regenerative medicine applications.

FED-BATCH PROCESS OUTPERFORMS BATCH CULTURE IN BIOREACTOR

• Fed-Batch process shows a distinct advantage on final cell yield and mediaproductivity over Batch process. (‘*’ indicates statistical significance between Fed-Batch and Batch systems at Day 5 and Day 6 (p<0.05)). Comparison study wasperformed in scaled down 0.1L bioreactors.

• Therefore, a cell expansion process was developed in which hMSCs are seeded ontomicrocarriers in a bioreactor on day 0, fed with RoosterReplenish on day 3, andharvested on day 5 (depicted below).

PROCESS FLOW OF hMSC EXPANSION IN BIOREACTORS

• hMSC cultures were sampled and monitored for cell growth on Corning® Synthemax®II microcarriers on day 1, 3, & 4 of bioreactor culture in the 0.1L system, while the 3L,15L and 50L systems were sampled on day 1, 2, 3, & 4 of bioreactor culture.

• After 1 day, cells attached to microcarriers were observed in all the bioreactorsystems, with increases in cell number and aggregation after 3 and 5 days of culture.

GROWTH PROFILE OF hMSCs

• Cell counts were performed daily to determine the cell densities and growth profilesfor each bioreactor system.

• The small (0.1L), development (3L), pilot (15L) and production (50L) scale bioreactorsystems resulted in similar growth kinetics, with all achieving a minimum of 0.5Mcells/ml within 4 - 5 days of culture among different donors.

• Greater final cell densities observed at the larger bioreactor scales suggests a moreefficient process at the larger scales, possibly due to better environmental controls.

• Furthermore, these results demonstrate the scalability of the bioreactor process tomeet potential clinical and commercial demand.

www.roosterbio.com

BIOCHEMICAL PROFILE OF MEDIA

• Concentration of glucose and glutamine nutrients were maintained at desired levelsthroughout culture to support cell expansion in all bioreactor scales.

• Waste accumulation, as measured by lactate and ammonia concentration, alsoremained similar across bioreactor scales.

• Overall, biochemical profiles were similar regardless of bioreactor volume, indicatingthat similar strategies can be used across bioreactor systems to control nutrients andwaste in the media.

• Cell counts were performed after in-vessel dissociation to determine the “CellDensity at Harvest” for each bioreactor system.

• Cell detachment from microcarriers was accomplished through agitation of thebioreactor wheel in the presence of an enzymatic cell dissociation reagent.

• Efficient removal of microcarriers from the bulk cell solution was accomplished bypassing through a filtration device.

• ǂ Parameters for downstream processes in the 50L scale will be further optimized.

CRITICAL QUALITY ATTRIBUTES AFTER 50L BIOREACTOR CULTURE & HARVEST

(G) IDO ACTIVITY(F) CYTOKINE SECRETION PROFILE

(A) OSTEOGENESIS (B) ADIPOGENESIS (C) CHONDROGENESIS

(D) CELL SURFACE MARKER EXPRESSION (E) POST-HARVESTEXPANSION IN 2D

• XF hMSCs harvested from the 50L bioreactor maintained their phenotypic properties,including tri-lineage differentiation potential to (A) osteogenic, (B) adipogenic, and(C) chondrogenic lineages; (D) classic surface protein expression; and (E) typical MSCmorphology after recovery from the bioreactor.

• Functional properties of XF hMSCs expanded in bioreactors were maintained andcomparable to 2D controls in terms of levels of (F) secreted angiogenic cytokines and(G) inducible indoleamine 2,3-dioxygenase (IDO) activity when stimulated withinterferon-gamma (IFNᵧ).

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Donor 1 Expansion in Different Bioreactor Scales

50L

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Donor 1

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Bioreactor Scale Cell Density at Harvest Total Cell Yield

0.1L (n = 3) 615,000 cells/ml 61.5 Million cells

3L (n = 3) 504,000 cells/ml 1.5 Billion cells

15L (n = 3) 664,000 cells/ml 10 Billion cells

50L (n = 3) 635,000 cells/ml 31 Billion cells

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DOWNSTREAM MANUFACTURING OPERATIONS

Bioreactor Cell / Microcarrier Separation Device

Closed-SystemCentrifugation

Aseptic Vial

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CELL DENSITIES & POST-HARVEST YIELDS

FGF HGF IL-8 TIMP-1 TMP-2 VEGF100

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2D Cell Stock Generation 3D Bioreactor Culture

CD14 CD34 CD45 CD73 CD90 CD105 CD166

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