TIPS: Titerless Infected-Cells Preservation and Scale-Up

Wasilko, David J.; Lee, S. Edward

doi:10.12665/j53.wasilkolee

Cited by 37 publications

(24 citation statements)

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“…Sf9 cells infected with His6-ENLYFQGA-NPC1(23-252)(N70Q/N122Q/N185Q) baculovirus (Wasilko and Lee, 2006) were used to infect Hi-5 cells at 1×10 6 cells/ml in Excel-405 medium. After incubation for 96 hr at 27°C, cells were pelleted by centrifugation, and the medium was concentrated by tangential flow filtration and exchanged into buffer A.…”

Section: Methodsmentioning

confidence: 99%

Structure of N-Terminal Domain of NPC1 Reveals Distinct Subdomains for Binding and Transfer of Cholesterol

Kwon

Abi-Mosleh

Wang

et al. 2009

Cell

605

703

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SUMMARY LDL delivers cholesterol to lysosomes by receptor-mediated endocytosis. Exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its isooctyl side chain buried and its 3ß-hydroxyl exposed. Here, we describe high-resolution structures of the N-terminal domain (NTD) of NPC1 and complexes with cholesterol and 25-hydroxycholesterol. NPC1(NTD) binds cholesterol in an orientation opposite to NPC2: 3ß-hydroxyl buried and isooctyl side chain exposed. Cholesterol transfer from NPC2 to NPC1(NTD) requires reorientation of a helical subdomain in NPC1(NTD), enlarging the opening for cholesterol entry. NPC1 with point mutations in this subdomain (distinct from the binding subdomain) cannot accept cholesterol from NPC2 and cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. We propose a working model: after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer lysosomal membranes.

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Section: Methodsmentioning

confidence: 99%

Structure of N-Terminal Domain of NPC1 Reveals Distinct Subdomains for Binding and Transfer of Cholesterol

Kwon

Abi-Mosleh

Wang

et al. 2009

Cell

605

703

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“…Baculovirus generation was accomplished using the Bac-to-Bac baculovirus generation system (Life Technologies). Flag-His 6 -TEV-IRE1a (547-977) protein expression in baculovirusinfected insect cells was accomplished following established procedures (Wasilko and Lee, 2006). Briefly, a proprietary Sf9 insect cell line was grown to the early log phase, infected with 1 Â 10 7 baculovirusinfected insect cells/10 l culture, and incubated at 27°C.…”

Section: Methodsmentioning

confidence: 99%

Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1αEndoribonuclease Activity

et al. 2015

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Activation of the inositol-requiring enzyme-1 alpha (IRE1a) protein caused by endoplasmic reticulum stress results in the homodimerization of the N-terminal endoplasmic reticulum luminal domains, autophosphorylation of the cytoplasmic kinase domains, and conformational changes to the cytoplasmic endoribonuclease (RNase) domains, which render them functional and can lead to the splicing of X-box binding protein 1 (XBP 1) mRNA. Herein, we report the first crystal structures of the cytoplasmic portion of a human phosphorylated IRE1a dimer in complex with (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide, a novel, IRE1a-selective kinase inhibitor, and staurosporine, a broad spectrum kinase inhibitor. (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide inhibits both the kinase and RNase activities of IRE1a. The inhibitor interacts with the catalytic residues Lys599 and Glu612 and displaces the kinase activation loop to the DFG-out conformation. Inactivation of IRE1a RNase activity appears to be caused by a conformational change, whereby the aC helix is displaced, resulting in the rearrangement of the kinase domain-dimer interface and a rotation of the RNase domains away from each other. In contrast, staurosporine binds at the ATP-binding site of IRE1a, resulting in a dimer consistent with RNase active yeast Ire1 dimers. Activation of IRE1a RNase activity appears to be promoted by a network of hydrogen bond interactions between highly conserved residues across the RNase dimer interface that place key catalytic residues poised for reaction. These data implicate that the intermolecular interactions between conserved residues in the RNase domain are required for activity, and that the disruption of these interactions can be achieved pharmacologically by small molecule kinase domain inhibitors.

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“…This process, previously described for recombinant protein production, is referred to as ''titerless infected-cell preservation and scale-up'' (TIPS) (Wasilko and Lee, 2006;Wasilko et al, 2009). The BIIC aliquots are cryopreserved late in infection, but before viability diminishes.…”

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confidence: 99%

Reproducible High Yields of Recombinant Adeno-Associated Virus Produced Using Invertebrate Cells in 0.02- to 200-Liter Cultures

Cecchini

Virág²,

Kotin³

2011

Human Gene Therapy

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The large amounts of recombinant adeno-associated virus (rAAV) vector needed for clinical trials and eventual commercialization require robust, economical, reproducible, and scalable production processes compatible with current good manufacturing practice. rAAV produced using baculovirus and insect cells satisfies these conditions; however, recovering rAAV particles from 200-liter bioreactors is more complicated than bench-scale vector preparations. Using a variety of processing media, we developed a reliable and routine downstream procedure for rAAV production that is scalable from 0.02-to 200-liter cultures. To facilitate the upstream process, we adapted the titerless infected-cell preservation and scale-up process for rAAV production. Single-use aliquots of cryopreserved baculovirus-infected insect cells (BIIC) are thawed and added to the suspension culture to achieve the desired ratio of BIIC to rAAV-producer cells. By using conditions established with small-scale cultures, rAAV was produced in larger volume cultures. Strikingly consistent rAAV yields were attained in cultures ranging from 10 liters to 200 liters. Based on the final yield, each cell produced 18,000 AE 6,800 particles of purified rAAV in 10-, 20-, 100-, and 200-liter cultures. Thus, with an average cell density of 4.32Â10 6 cells/ml, !10 16 purified rAAV particles are produced from 100 to 200 liters. The downstream process resulted in about 20% recovery estimated from comparing the quantities of capsid protein antigen in the crude bioreactor material and in the final, purified product. The ease and reproducibility of rAAV production in 200-liter bioreactors suggest that the limit has not been reached, and 500-liter productions are planned.

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TIPS: Titerless Infected-Cells Preservation and Scale-Up

Cited by 37 publications

References 0 publications

Structure of N-Terminal Domain of NPC1 Reveals Distinct Subdomains for Binding and Transfer of Cholesterol

Structure of N-Terminal Domain of NPC1 Reveals Distinct Subdomains for Binding and Transfer of Cholesterol

Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1αEndoribonuclease Activity

Reproducible High Yields of Recombinant Adeno-Associated Virus Produced Using Invertebrate Cells in 0.02- to 200-Liter Cultures

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