Improvement of clarification process in monoclonal antibody manufacturing: effect of flocculation agent concentration on the filterability of cell culture supernatant

Sara Mafirad,1,* Arman goudarzi,2 Hassan morsal,3 Hamideh aghajani,4

1. Manufacturing department, Aryogen Pharmed Co., Makhsous Tehran-Karaj highway, 24th Km, Karaj, Iran
2. Manufacturing department, Aryogen Pharmed Co., Makhsous Tehran-Karaj highway, 24th Km, Karaj, Iran
3. Manufacturing department, Aryogen Pharmed Co., Makhsous Tehran-Karaj highway, 24th Km, Karaj, Iran
4. Manufacturing department, Aryogen Pharmed Co., Makhsous Tehran-Karaj highway, 24th Km, Karaj, Iran

Abstract


Introduction

The clarification of cell culture harvest can be daunting task due to the large volumes of harvest processed from modern production batch bioreactors that requires primary and often secondary clarification prior to application to a capture chromatography operation. flocculation has been shown to be effective in providing an aiding solution for clarification of processing high density cell culture harvests. flocculation of negatively charged compounds found in cell culture with cationic polymers (e.g., polydiallyldimethylammonium chloride (pdadmac), polyamines, polyaminoacids, polyacrylamides,and chitosan) takes place through ionic interactions that result in the neutralized particle falling out of solution. multiple antibody feed streams treated with addition of flocculation agent and subsequently filtered resulted in improved removal of cells and colloids, increased clarification throughput, efficient reduction of dna and high process yield (tomic et al., 2015). the polymer amount is dependent on the cell type or size and considerations for residual polymer should be weighed in addition to filtrate quality and impurity clearance to minimize the risk of any harm for patients (mcnerney et al., 2015). in this study, effect of flocculation agent concentration on the turbidity and filterability of the centrate supernatant of cell culture has been investigated

Methods

Cell broth was produced by aryogen pharmed proprietary batches or perfusion cell culture processes using cho cells that expressed igg monoclonal antibodies. filtration throughput was determined using 0.22 µm syring filters (sartorius, minisart, high flow syringe filters, germany). flocculation agent were obtained from merck. samples were prepared after adding the flocculation polymer with the indicated concentrations (0.002% - 0.05%) to the 50 ml falcon tubes and let to mix for 20 min in the shaker incubator at ambient temperature with an rpm of about 70. then, the flocculated particles were separated from the supernatant via centrifuge (hettich bench top centrifuge universal 320, germany) at 1000 rpm for 5 minutes. to investigate the effect on the filtration step with 0.22 μm filter, specified amount of centrate supernatant was passed through a syringe filter. the filtrated volume were recorded. the tests were done for 5 different batch of manufacturing.

Results

For investigation of the flocculant concentration influence on clarification process of cell culture harvest, centrated supernatant turbidity and filterability changes verses the polymer dosage were studied. from figure1, it is revealed that with increase of flocculation agent concentration from 0.002 to 0.01% the turbidity of centrate has been increased in comparison with the blank centrate. but for higher concentration the turbidity decreasing were obtained. with the polymer concentrations of 0.03% the turbidity decline significantly. the polymer dosage increase from 0.03% to 0.05%, revealed very broad clearance efficiency. the same results were reported by burgstaller et al. (2017). at the concentrations lower than 0.01%, the polymer addition is not sufficient to build large flock structures for sedimentation, but only it causes to increase the particle size and consequently result in higher turbidity centrates. figure 1. turbidity of the centrates vs flocculation agent percentage this result shows the necessity of the flocculant concentration optimization in order to avoid any adverse effect on the depth clarification and filtration process. for investigation of flocculant concentration on filterability of centrate, an index of v* were specified as follow: v_*=(filtrate volume of centrate (with flocculant)(ml))/(filtrate volume of the blank centrate (ml)) results is shown for all the batches in figure 2. the higher value of the index. the higher filterability. figure 2. index of filterability changes with the flocculant concentration increase regarding figure2, index of filterability stepped up due to the flocculant concentration change from 0.02% to 0.03%. therefore 0.03% would be the optimum concentration of needed flocculant in this study.

Conclusion

Results showed the necessity of the flocculant concentration optimization in order to avoid any adverse effect on the depth clarification and filtration process. it is revealed that at the polymer concentrations lower than 0.01%, increase of concentration, result in the centrate turbidity increase. also regarding the filterability index, at optimum concentration of needed flocculant (0.03%), filterability and turbidity of the supernatant were considerably improved.

Keywords

Flocculation , cell culture, clarification, turbidity, filterability.