EVALUATION OF MACROPOROUS AND MICROPOROUS CARRIERS FOR CHO-K1 CELL GROWTH AND MAB PRODUCTION

Author* RODRIGUES, M.E.; COSTA, A.R. Supervisors: Oliveira, R.; Azeredo, J.; Henriques, M.
University of Minho School of Engineering Center of Biological Engineering
* elisarodrigues@deb.uminho.pt

Introduction
Large-scale biopharmaceutical production commonly relies on suspension cell cultures that provide higher yields than adherent cultures. However, most mammalian cells grow adherently and need to be adapted to suspension, which is not always simple or feasible. Microcarrier culture introduces new possibilities and allows the practical high yield culture of anchorage-dependent cells in suspension systems. In these systems, cells grow as monolayers on the surface of the microcarriers, which are usually in suspension in the culture medium by gentle rocking. The aim of the study was to evaluate, compare and optimize the use of microcarriers for the growth and antibody production by CHOK1 cells.

Results
INITIAL ADHESION TO MICROCARRIERS
2x105 cells/ml
400000 350000 300000 250000 200000 150000 100000 50000 0 0 1 2 3 4 12000000 10000000 8000000 6000000 4000000 2000000 0

CELL PROLIFERATION IN MICROCARRIERS

2x105 cells/ml

MONOCLONAL ANTIBODY PRODUCTION IN MICROCARRIERS

AVERAGE PRODUCTIVITY / (pg/cell/day) Cytodex 3 CultiSpher S TOTAL PRODUCTION / (g/ml)

C cells adhered / (cells/ml)

C cells adhered / (cells/ml)

CultiSpher S 13.60 11.78 14.64 17.84

Cytodex 3 14.77 11.16 15.94 12.43

CultiSpher S Pulse Cytodex 3

Time / (h)
5No 6 differences

CultiSpher S No differences Cytodex 3

0 5 10 15 20 25 Continuous

C1 Pulse Continuous C2 Pulse Continuous

2.11 1.16 1.61 2.99

1.85 0.32 0.70 0.57

Methods

Time / (days)

CHO-K1 cells producing monoclonal antibody (mAb) as well as the macroporous CultiSpher S and microporous Cytodex 3 microcarriers were used and compared. Microcarrier cultures of 5 ml were performed at 37C, 5% CO2, in 50 ml tubes. Medium was changed every day and samples were taken for microcarrier and cell counting, and antibody quantification by ELISA. The set of conditions described in scheme 1 was analysed.
400000 350000 300000 250000 200000 150000 100000 50000 0 0 1 2

4x105 cells/ml
12000000 10000000 8000000 6000000 4000000 2000000 0

4x105 cells/ml

Pulse favours production for C1, and continuous favours production for C2

C cells adhered / (cells/ml)

C cells adhered / (cells/ml)

CultiSpher S Continuous Cytodex 3

3 4 5

CultiSpher S No differences Cytodex 3

0 5 10 15 20

Higher total production for CultiSpher with C2 and continuous rocking

Time / (h) Pulse

400000 350000 300000 250000 200000 150000 100000 50000 0 0 1 2 3 4 5

6 Continuous

CultiSpher S with better productivity (less cells achieve the same production)

Time / (days)

25 Pulse

Pulse
12 10 8 6 4 2 0

Conclusions

C cells adhered / (cells/ml)

C cells adhered / (cells/ml) C cells adhered / (cells/ml)

CultiSpher S C1 Cytodex 3 C2 6
Time / (h) Continuous

CultiSpher S No differences Cytodex 3

0 5 10 15 20 25 C2

Microcarrier cultures proved to be a viable alternative to suspended cultures for the growth and antibody production by CHO-K1 cells. During initial cell adhesion, the use of higher inoculum concentration is particularly favorable if continuous rocking is used. For both microcarriers, the majority of cell adhesion occurs within the first 3 hours. In general, higher levels of initial adhesion are obtained with Cytodex 3. The higher mAb productivity and total production are achieved with CultiSpher S cultured with 4x105 cells/ml and continuous rocking methodology. Cytodex 3 is recommended for purposes of cell growth (provides higher levels of cell adhesion and proliferation), while CultiSpher S is indicated for purposes of production (provides higher productivities).

Time / (days)

Continuous
12000000 10000000 8000000 6000000 4000000 2000000 0 0 5 10 15 20

400000 350000 300000 250000 200000 150000 100000 50000 0 0 1 2 3 4 5

C cells adhered / (cells/ml)

CultiSpher S C2 Cytodex 3 C26

Time / (h)

CultiSpher S No differences Cytodex 3

25 C1

4x105 cell/ml (C2) CultiSpher S - 1 g/L Cytodex 3 - 3 g/L Continuous (60 rpm) Pulse (60 rpm, 1 min each hour)

Cell proliferation

INOCULUM CELL CONCENTRATION MICROCARRIER CONCENTRATION ROCKING METHODOLOGY

2x105 cell/ml (C1)

Initial adhesion (6 hours)

Time / (days)

Better combination for initial cell adhesion: C2 continuous. For both CultiSpher S and Cytodex 3, it provides the higher number of cells adhered

Cytodex 3 has higher cell proliferation than CultiSpher S Cytodex 3 has higher durability than CultiSpher S, which starts to desintegrate after two weeks

Scheme 1. Set of conditions for CultiSpher S and Cytodex 3 cultures.

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