Debottlenecking Manufacturing Capacity: Initiating cell culture manufacturing campaigns using seed train cryopreserved in a disposable bag. Shahid Rameez, Ph.D. KBI Biopharma Inc. Research Triangle Park, NC 251 st ACS National Meeting & Exposition, San Diego, California, March 13, 2016.
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Debottlenecking Manufacturing Capacity: Initiating cell culture manufacturing campaigns using seed train cryopreserved in a disposable bag
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251st ACS National Meeting & Exposition, San Diego, California, March 13, 2016.
Background
A key bottleneck for mammalian cell culture productivity is the extended duration of the process with inoculum seed train and production culture stretching between 4-6 weeks in duration.
Introducing flexibility in scheduling and execution of cell culture manufacturing campaigns with via a reduction in process duration can be a key strategy for maximizing facility utilization and facilitating the progression of multiple therapeutics to clinical trials.
In this work, we investigated the initiation of CHO cell culture production runs using seed cultures cryopreserved in large disposable bags.
Major Challenges/Questions
How do we cultivate cells to high cell densities without compromising cell growth characteristics?
In what do we cryofreeze the cells in larger volumes?
Will the cells sustain when cryofrozen in large volumes/densities?
Will the cell growth, productivity and product quality match to the traditional seed trains?
What will the long term storage at larger volumes have any negative effect on the cells?
Overview
This presentation focuses on outlining a development approach which involves cultivating cells to high density prior to cryopreservation using two approaches:
(1) ATF cell retention system, which separates spent medium from cells using a hollow fiber filter.
(2) kSep® system, which harvests cells as a concentrated fluidized bed under a continuous flow of media using the balance of centrifugal and fluid flow forces.
The case studies presented here will illustrate the use of the seed train
cryopreserved in disposable bags for initiation of CHO cell culture production runs for cell lines producing mAbs.
Bioreactor Processes
Batch Fed-Batch Continuous
Cell Retention Device
Perfusion
Alternating Tangential Flow (ATF) System Filtration based on Alternating Tangential Flow (ATF) using a diaphragm pump.
Exhaust(E) Cycle
Pressure (P) Cycle
ATF Cleaning Action
Back-flush Action
kSep® System kSep Systems’ utilize patented kSep® technology that either harvest cells as
product or discard cells as by-product during manufacturing.
Through the balance of centrifugal and fluid flow forces, the kSep® retains cells as a concentrated fluidized bed under a continuous flow of media.
kSep® System
Key Features: Handles low to high cell density cultures (0.05 - 100 million cells/mL).
Provides option to selectively remove small particulate impurities, e.g. cell debris, leachable from plastic and components of freezing media.
Imparts low shear on cells and maintains a healthy environment to sustain cell viability.
Closed system with single-use class VI product contact surfaces
Aim
Passage 1 Passage 2 Passage 3 Passage 4
3 day passages (Shake Flasks)
2000L
Bioreactor
Conventional Process
3 day passage (Wave)
14 - 16 day Production
2.0-2.5 weeks
2.0-2.5 weeks
Modified Process
Cryofrozen Cell Disposable Bag
200L
Bioreactor
3 day passage (N-1)
10 - 16 day Production
1.5-2.5 weeks
2000L
Bioreactor
MCB Vial Thaw
Passage 1 Passage 2
3 day passages (Shake Flasks)
Work Flow 1: Cryofrozen cell disposable bags using ATF cell retention system
7- 10 day culture
Cryofrozen Cell Disposable Bag
Passage 3 Passage 4 50L
Bioreactor
Work Flow 2: Cryofrozen cell disposable bags using kSep® system to harvest cells at concentrated levels.
MCB Vial Thaw
Passage 1 Passage 2
3 day passages (Shake Flasks) 3 day passage
Passage 3 Passage 4
3 day passage
Cryofrozen Cell
Disposable Bag kSep
Generating Frozen Inoc.
200L
Bioreactor
Results: ATF to Increase Cell density.
Cell densities greater than 100 million cells/mL with high viabilities were achieved with ATF.
Note: The exponential growth observed in ATF and achieving high density can be utilized at N-1 stage. These high density cultures can be subsequently used to inoculate production bioreactors at high seed density (5-10 million cells/mL) to start fed-batch production cultures. This strategy can significantly shorten the culture duration. (Approach used by many groups in the industry)
Results: kSep® to Increase Cell Density.
Cell densities greater than 60 million cells/mL and high cell viabilities were achieved with kSep. kSep provides option to selectively remove dead cells and small particulate impurities like cell debris which in turn help in increasing culture viability.
Note: kSep, similar to ATF, can be used to achieve high density at N-1 stage and inoculate production bioreactors at high seed density to shorten fed-batch production culture duration.
Seed Culture
VCD: 6.04 million cells/mL
Viability: 86%
High Density Culture
VCD: 63.10 million cells/mL
Viability: 91%
Seed Culture
VCD: 5.0 million cells/mL
Viability: >95%
High Density Culture
VCD: 25 million cells/mL
Viability: >95%
Results: Generation Of Cryofrozen Bags.
The bags and bag accessories are tested for Liquid Nitrogen Storage (> -190oC)
High density Cultures (ATF or kSep)
(Suspended in Freezing Medium)
Cryofreeze Compatible Disposable Bags
30 - 300mL @ 20-50 million cells/mL
-80oC Overnight Storage
Liquid Nitrogen Longterm Storage
Results: Cryofrozen Seed Train Evaluation
Similarly, major metabolite profiles, productivity and critical product quality attributes compared well to the production runs with conventional seed train process initiated from cell bank ampoule.
Note: The cell morphology of Cryofrozen cells from disposable bags (circularity, average diameter, size distribution) compared very well with conventional cells from cell vial ampoules.
Ongoing and Future Experiments
Improving the workflow for the banking of cells in the CryoBags.
Developing assays for CryoBag characterization (Mitochondrial potential assays and mitochondrial transition pore assays). Evaluate more molecules/cell lines .
Acknowledgements
Joe McMahon (President and CEO)
Abhinav Shukla, Ph.D. (Senior VP, Process Development and Manufacturing)
Sigma Mostafa, Ph.D. (VP, Process Development)
Lab members Srivatsan Gopalakrishnan Carl Zhang Derek Ryan, Ph.D. (and his group)