Investigation and optimization of appropriate condition for rfvii activation

Davood Yavari,1 Samaneh bayati,2 Rasoul garousi,3 Fereshteh shams abadi,4 Somayeh marjani nia,5 Neda abadian,6,*

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
6. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran

Abstract


Introduction

Rfvii is a 50 kd serine protease which hydrolysis the sequence between arg152-ileu153 bond produces a 2-chain molecule composed of disulfide-linked polypeptides about 20 and 30 kd that this cleavage is necessary to produce active form of rfvii (rfviia) [1]. this hydrolysis has not been stopped after auto-activation because of the cleavages at other sites which is known as auto-degradation. the degraded form of protein will be increased when the rfvii activation takes more time. as hence, optimization of activation time before protein degradation is a critical concern in downstream processing of rfvii. in this study the effect of different parameters including the concentration of rfvii and calcium ion, selection an appropriate temperature for activation and determination of optimum insulation time of rfvii after using positively charged surfaces were investigated.

Methods

In this study, the activation of rfvii with concentration of 1.5, 3, 6 and 12 mg/ml were investigated in presence of different concentration of cacl2 (5, 20 and 50 mm). in the following, to further investigation about the effect of positively charged surfaces of media, 6 ml of q-sepharose media was packed onto xk16/20 column and the target protein with 12 mg/ml of media binding capacity was loaded onto the column followed by elution step using 50 mm cacl2. the effect of two contact times, 6 and 18 h, as well as two different temperatures, 4 & 25 °c, were explored under this condition. the trend of activation and degraded form were evaluated by sds-page and rp-hplc respectively.

Results

The results showed that a reverse correlation between in-activation time and concentration of desired protein. non-active form in 0.5 mg/ml concentration of rfvii was still more than 50 % after 48 h while more than 90 % of protein was activated in 3, 6 and 12 mg/ml after 48, 24 and 8 h respectively. in addition, the results cleared that the activation time wasn’t changed with increasing the concentration of cacl2. in other words, although ca2+ concentration was increased 10 folds from 5 to 50 mm, activation time in different concentration of rfvii was constant. the lowest incubation time was about 8 h when rfvii with concentration of 12 mg/ml was incubated in presence of 50 mm cacl2. in the following, the results cleared that the presence of positively charged surfaces, q-sepharose media, was led to decrease the activation time from 8 h to about 6 h compare to the previous experiment when ca2+ was directly added. in addition, the activation time was decreased with increasing the time of exposure of rfvii with positive charge of q-sepharose media’s surface. furthermore, the results of contact time showed that although the activation of protein after 18 h was 3 folds more than 6 h, this time increasing was caused to slight enhancement in degraded forms. in the following, further investigation about the effect of temperature on activation showed that there were no significant difference in activated forms of the samples which were kept in cold room and room temperature.

Conclusion

Overall, the results showed that rfvii activation is more affected by the concentration of target protein rather than the other parameters investigated in this study. although, the presence of cacl2 is crucial for activation but using different concentration of cacl2 doesn’t have significant effect. as the results, optimum activation time was obtained about 8 h with concentration of 12 mg/ml of rfvii. also, the results suggests that the contact time of target protein to media is another parameter that should be considered during the process to achieve the lowest activation time and prevent the formation of degraded forms. moreover, the results showed that the temperature has no effect on this process.

Keywords

Activation, cacl2, q-sepharose, rfvii