title: Use of hollow fiber tangential flow filtration for the recovery and concentration of HIV virus-like particles produced in insect cells.
abstract: 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 μm 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 2L of starting cell suspension within 6h 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.
authors.affiliation: Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, United States. Electronic address: negretea@mail.nih.gov.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Negrete
authors.forename: Amrita
authors.initials: A
authors.lastname: Pai
authors.forename: Joseph
authors.initials: J
authors.lastname: Shiloach
mesh_terms: D016915:AIDS Vaccines / Q000302:isolation & purification*; D000818:Animals; D005374:Filtration / Q000379:methods*; D015488:HIV Antigens / Q000096:biosynthesis*; D007313:Insecta; D011994:Recombinant Proteins / Q000096:biosynthesis; D058425:Vaccines, Virus-Like Particle
chemical_list: D016915:AIDS Vaccines; D015488:HIV Antigens; D011994:Recombinant Proteins; D058425:Vaccines, Virus-Like Particle
keywords: Concentration; Hollow fiber; Insect cells; Recovery; Tangential flow filtration; Virus-like particles