Optimization of agitation speed to improve monoclonal antibody production through the control of lactate accumulation

Raziyeh Arjmand,1 Sepideh samavat,2 Fatemeh ashouri,3 Aref hemmati,4 Shaian maleknia,5,*

1. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
2. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
3. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
4. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
5. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran

Abstract


Introduction

Monoclonal antibodies (mab) are an important class of biotherapeutics drugs for the treatment of cancer, autoimmunity and inflammatory diseases. chinese hamster ovary (cho) cells are commonly used mammalian hosts for the commercial production of therapeutic proteins. optimization of cell culture bioprocess is important to achieve high protein expression levels. accumulation of by-products in cho cell cultures is a concern because of its negative effects on cell growth and productivity. lactate is regarded as an inhibitory by-product that leads to intracellular ph changes, disturbance of electrochemical gradients and the inhibition of enzymatic reactions. in recent years, a number of methods have been developed to minimize lactic acid.

Methods

A recombinant chinese hamster ovary cell line producing a human mab, was cultured in chemically defined medium which was supplemented with 6 mm l-gln. fed-batch experiments were performed in 500 ml shake flasks with 100 ml working volume with initial seeding density of 0.6×106 viable cells/ml). three conditions were designed with different agitation speeds (a: 70-90 rpm, b: 80-100 rpm and c: 100-150 rpm) to evaluate the effect of different agitation speed on protein production. cells were incubated at 37 °c with 5% co2, and temperature shift from 37 °c to 32 °c on day 5. f-2a and f-2b feeds were added respectively from day 2 till day 13 (3% v/v f-2a and 0.3% v/v f-2b). cell density and viability were measured by the trypan blue exclusion method. ph value, osmolality, glucose and lactate concentrations were monitored daily by using bioprofile 400 analyzer.

Results

Results showed that lactate concentration was declined significantly when agitation speed increased up to 150 rpm and ph value was increased slightly. maximum viable cell density was achieved in condition b and c and as expected maximum protein production was achieved in these two conditions. maximum productivity increased 1.4 fold compared to control condition.

Conclusion

This study illustrate that aeration is one of the main parameters to control the lactate accumulation. optimization of agitation speed resulted in decreasing the lactate concentration in culture and consequently results in maximum productivity.

Keywords

Monoclonal antibody, cho cell, by-product, lactic acid, aeration