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

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)

Thanks..

Questions?