期刊
JOURNAL OF VIROLOGICAL METHODS
卷 195, 期 -, 页码 240-246出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jviromet.2013.10.017
关键词
Virus-like particles; Recovery; Concentration; Insect cells; Hollow fiber; Tangential flow filtration
资金
- National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health
Attenuated viruses, inactivated viruses and virus like particles (VLPs) are known to be efficient vaccines partially due to their particulate structure. A potential HIV vaccine candidate engineered as a VLP (HIV gag-VLP) and produced in insect cells is currently under preclinical trials demanding large amounts. Due to their extreme fragility and sensitivity to shear forces the recovery and concentration of these extracellular enveloped particles of approximately 120 nm in size is challenging. The current bench scale gradient ultracentrifugation and precipitation methods have been found unsuitable for larger scale processes. In this study a two-step tangential flow filtration (TFF) process using hollow fibers was developed for the clarification and concentration of HIV gag-VLPs. The first step is microfiltration for cell removal and the second step is ultrafiltration for concentrating the HIV gag-VLPs. The chosen parameters for the microfiltration step were hollow fiber membranes of 0.45 mu m cut off 5000 s(-1) shear force and a flux of 10 LMH. The chosen parameters for the ultrafiltration step were a 500 kDa cut off membrane, 6000 s(-1) shear force and a trans-membrane pressure (TMP) of 1.25 bar. The utilization of these parameters provided with concentrated HIV-gag VLPs from 2 L of starting cell suspension within 6 h of processing time. These downstream processing conditions are extremely valuable for the further large-scale purification process development for-HIV gag-VLPs and other particulate bioproducts. Published by Elsevier B.V.
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