BACKGROUND
lv vectors provide powerful tools for transgene delivery into dividing as well as nondividing cells and for long-term transgene expression in target cells in vitro and in vivo.
currently, the use of lv vectors is commonplace and applications in the fields of neuroscience, hematology, developmental biology, stem cell biology and transgenesis have emerged  <cit> .
lv vectors are also being pursued in a number of clinical trials .
despite significant progress, the production and concentration of high-titer, high-quality lv vectors for preclinical studies in animal models is still cumbersome and costly  <cit> .
the production of lv vectors is typically carried out using transient transfection approaches involving tissue culture dishes or flasks  <cit> , cell factories  <cit> , or stirred-tank bioreactors  <cit> .
these protocols are cumbersome to scale up  or technically challenging , preventing their routine use in a standard laboratory setting.
typical lv vector titers involving the vesicular stomatitis virus  g glycoprotein range from  <dig> to  <dig> tu/ml  <cit> .
higher titers can be achieved by physical concentration  <cit> , including ultracentrifugation  <cit> , or filtration approaches such as ultrafiltration  <cit> , and diafiltration  <cit> .
vector production for large-scale in vivo applications in animal models requiring high-titer lv vector stocks is challenging due to the lack of simple procedures allowing rapid processing of large volumes of lv vector-containing cell culture supernatants.
the traditional ultracentrifugation-based methods are limited in terms of their capacity to handle large volumes, thus making this procedure extremely tedious.
filtration approaches such as diafiltration are well suited for processing large volumes of vector supernatants.
however, they are difficult to implement in a standard laboratory setting.
thus, there is an emerging need for simple and less laborious procedures that result in a rapid reduction of the volume of the cell culture supernatant to be processed without the need for a centrifugation step.
one problem with the centrifugation and filtration-based methods outlined above is that cell-derived components are concentrated along with the vector particles.
these have the potential to cause immune and inflammatory responses  <cit> .
for example, concentrated vsv-g-pseudotyped lv vector preparations were shown to be contaminated with tubovesicular structures of cellular origin which carried nucleic acids, including the plasmid dnas that were used to generate the lv vector stocks.
dna carried by these tubovesicular structures acted as a stimulus for innate antiviral responses, triggering toll-like receptor  <dig> and inducing alpha/beta interferon production  <cit> .
thus, additional steps including chromatography-based methods such as anion exchange chromatography are needed in order to reduce host cell and cell culture-derived contaminants from lv vector preparations.
methods based on anion exchange column chromatography of lv vectors pseudotyped with vsv-g  <cit>  or the baculovirus gp <dig> glycoprotein were previously described  <cit> .
however, the yields and purity of the lv vector stocks obtained in this way were not reported.
in an attempt to simplify the production and concentration of lv vectors and to make this approach more reproducible and cost-effective for preclinical studies in animals, we have worked out a facile lv vector production system based on hyperflasks.
we also implemented a straightforward concentration procedure based on mustang q anion exchange membrane chromatography.
mustang q anion exchange-based chromatography protocols for concentrating/purifying lv vectors were previously reported  <cit> .
such vector preparations displayed reduced toxicity compared to vectors concentrated using ultracentrifugation  <cit> , as well as enhanced purity  <cit> .
RESULTS
simplified production of lentiviral vectors using hyperflasks
with a view toward improving high-titer lv vector production for preclinical studies in animals, we tested the usefulness of hyperflask vessels that have a total growth area of  <dig> cm <dig>  corresponding to ten standard t <dig> flasks.
hyperflasks consist of ten interconnected growth surfaces each containing a membrane pretreated to allow improved cell adherence.
the membrane is gas permeable, allowing exchange of oxygen and carbon dioxide through the base of the membrane, resulting in gas exposure to a large surface area within the flask  <cit> .
to test the usefulness of hyperflasks for lv vector production involving calcium phosphate-mediated transfection  <cit> , we compared the titers of lv vector stocks prepared side-by-side using either ten 150-cm <dig> dishes or a single hyperflask.
the data shown in table  <dig> represent the results of three independent productions each for the 150-cm <dig> dish system and the hyperflask system.
these data indicate that the titers of unconcentrated lv vectors produced using hyperflasks were up to  <dig>  ×  <dig> tu/ml while the titers of lv vectors produced in 150-cm <dig> dishes were lower, up to  <dig>  ×  <dig> tu/ml.
the total yields were up to  <dig>  ×  <dig> tu from ten 150-cm <dig> dishes and up to  <dig>  ×  <dig> tu for the hyperflask vessels.
this corresponds to a productivity of up to  <dig> ×  <dig> tu per cm <dig> for the 150-cm <dig> dishes.
for the hyperflask, the productivity was about 10-fold higher, up to  <dig>  ×  <dig> tu per cm <dig>  the higher productivity observed with hyperflasks may be related to better gas exchange during lv vector production.
overall, the 293t cells used for production appeared healthier and there was less cell debris from transfections carried out in hyperflaks compared to transfections carried out in 150-cm <dig> dishes .
we expect the hyperflask production protocol described here to be compatible with other dna transfection formats such as polyethylenimine-mediated transfection  <cit> .
ten 150-cm <dig> dishes  and one hyperflask vessel  were used for lv vector production.
productions were performed side-by-side .
vector titers were determined using hos cells.
data are expressed as the mean ± standard deviation .
simplified concentration of lentiviral vectors using mustang q acrodiscs
the concentration and purification of lv vector stocks on a large scale presents a significant bottleneck and methods allowing rapid processing of large volumes of lv vector-containing supernatants are needed.
chromatography-based methods appear to be particularly attractive in this regard  <cit> .
for example, methods to concentrate/purify lv vectors based on anion exchange or affinity chromatography have been established  <cit> .
recently, segura et al.
<cit>  described an approach involving heparin affinity chromatography to concentrate lv vector particles directly from clarified supernatants.
during this step, a recovery of 53% of infectious lv particles was achieved while 94% of the impurities were removed.
this strategy may ultimately result in vector stocks of improved purity, increased infectivity and reduced toxicity.
however, these approaches are cumbersome and difficult to implement in a standard laboratory setting.
we have recently described a facile membrane-based chromatography approach involving mustang q anion exchange chromatography cartridges  to purify adenoviral vectors  <cit> .
mustang q membranes resulted in high recoveries of infectious viral particles and allowed the processing of adenoviral vector particles from lysates in a fraction of the time required using the traditional cesium chloride-based ultracentrifugation method.
mustang q membranes include a matrix that has a high dynamic binding capacity for adenoviral vectors and is capable of withstanding high flow rates.
other benefits of mustang q membrane chromatography cartridges include higher peak resolution at faster flow rates as compared to traditional ion exchange resins.
also, they can easily be adapted to bench-scale work and they are syringe-adaptable.
finally, there is no need for an hplc setup or a column packing step.
in work with lv vectors carried out by us  <cit>  and by others  <cit> , mustang q-based cartridges were used to concentrate/purify lv vectors.
while these initial studies were encouraging, the capacity of mustang q membranes for crude lv vector supernatants was rather low  <cit> , possibly caused by cell-derived proteins, serum proteins, or contaminating nucleic acids that bound to mustang q membranes during vector capturing.
to determine the binding capacity of mustang q membranes for lv vectors produced in hyperflask vessels compared to 150-cm <dig> dishes, a capture study was conducted.
figure  <dig> shows a typical elution profile of hyperflask-produced lv vectors following capturing onto a  <dig> -ml mustang q acrodisc and elution using a gradient ranging from  <dig>  to  <dig>  m nacl.
tu were determined by facs analysis of transduced hos cells.
the results presented in table  <dig> show that the capacity of mustang q acrodiscs at 10% breakthrough for lv vector supernatants produced in 150-cm <dig> dishes was up to  <dig>  ×  <dig> tu per ml of membrane while the capacity of mustang q membranes at 10% breakthrough for hyperflask vessel-derived lv vector samples was up to  <dig>  ×  <dig> tu per ml of membrane.
this is consistent with the observation that lv vector supernatants produced using hyperflasks contained fewer cellular proteins and nucleic acids compared to supernatants produced in 150-cm <dig> dishes  that may have interfered with the binding of the lv vector particles.
to remove excess nacl, pooled mustang q fractions were subjected to a buffer exchange using sepharose cl-4b.
table  <dig> summarizes the recovery of lv vectors following processing of 500-ml aliquots of hyperflask vessel-derived supernatants.
up to  <dig>  ×  <dig> tu were obtained after mustang q chromatography and up to  <dig>  ×  <dig> tu after subsequent buffer exchange using sepharose cl-4b .
mustang q acrodiscs were challenged with 75-ml aliquots of vector-containing supernatants.
the crude vector supernatants were filtered using a  <dig>  μm filter prior to anion exchange chromatography on mustang q acrodiscs.
input corresponds to the total number of tu loaded.
unbound corresponds the total number of tu found in the flow-through.
bound represents input tu minus unbound tu.
recovered corresponds to the total number of tu following elution.
capacity corresponds to the total number of tu bound per ml of the mustang q membrane.
since the hyperflask-derived samples did not result in greater than 10% breakthrough, the upper limit of the capacity for these samples is not known.
titers were determined by facs using hos cells.
data from three independent experiments involving 150-cm <dig> dish-derived and hyperflask vessel-derived vector supernatants are shown.
the data are expressed as mean ± sd.
protein and dna concentrations of 150-cm <dig> dish-derived and hyperflask vessel-derived vector supernatants were determined using a qubit assay kit .
productions were performed side-by-side .
data are expressed as mean ± sd.
mustang q acrodiscs were challenged with 500-ml aliquots of hyperflask vessel-derived vector supernatants.
buffer exchange was carried out by sepharose cl-4b size exclusion chromatography as outlined under materials and methods.
titers were determined by facs using hos cells.
data from three independent productions are shown.
data are expressed as mean ± sd.
finally, we wanted to compare the titers and total yields of our concentrated lv vectors with those reported by other groups.
to do this, we carried out a side-by-side titer comparison of a hyperflask-derived lv vector stock concentrated by mustang q anion exchange chromatography using hos cells, 293t cells and hela cells.
table  <dig> shows that the titers of fresh  vector stocks were similar in hos cells and 293t cells and slightly lower in hela cells.
upon freezing and thawing of mustang q-concentrated vector stocks there was a 40% drop in vector titers on average .
freshly concentrated lv vector stocks were diluted and aliquots were used to transduce hos, 293t and hela cells as outlined under materials and methods.
titers were determined by facs .
data are expressed as mean ± sd.
overall, the titers obtained using the hyperflask system were as high or higher compared to those obtained using 150-cm <dig> dishes and ultracentrifugation-based concentration approaches  <cit> .
the yield  from a single hyperflask  was above  <dig> tu .
this is about two orders of magnitude higher than the yields reported by karolewski et al.
<cit>  who used a cell factory system , and at least one order of magnitude higher than the yields reported by geraerts et al.
<dig>  <cit>  who also used cell factories.
CONCLUSIONS
in conclusion, we present here a simple protocol for lv vector production and concentration involving hyperflasks in conjunction with mustang q anion exchange membrane cartridges.
these protocols are easy to implement in a standard laboratory setting and allow high-titer lv vector production without the need for multiple tissue culture dishes or flasks or ultracentrifugation-based concentration procedures.

 lv vectors provide powerful tools for transgene delivery into dividing as well as nondividing cells and for long-term transgene expression in target cells in vitro and in vivo. currently, the use of lv vectors is commonplace and applications in the fields of neuroscience, hematology, developmental biology, stem cell biology and transgenesis have emerged. lv vectors are also being pursued in a number of clinical trials. despite significant progress, the production and concentration of high-titer, high-quality lv vectors for preclinical studies in animal models is still cumbersome and costly. the production of lv vectors is typically carried out using transient transfection approaches involving tissue culture dishes or flasks, cell factories, or stirred-tank bioreactors. these protocols are cumbersome to scale up or technically challenging, preventing their routine use in a standard laboratory setting. typical lv vector titers involving the vesicular stomatitis virus g glycoprotein range from to tu/ml. higher titers can be achieved by physical concentration, including ultracentrifugation, or filtration approaches such as ultrafiltration, and diafiltration. vector production for large-scale in vivo applications in animal models requiring high-titer lv vector stocks is challenging due to the lack of simple procedures allowing rapid processing of large volumes of lv vector-containing cell culture supernatants. the traditional ultracentrifugation-based methods are limited in terms of their capacity to handle large volumes, thus making this procedure extremely tedious. filtration approaches such as diafiltration are well suited for processing large volumes of vector supernatants. however, they are difficult to implement in a standard laboratory setting. thus, there is an emerging need for simple and less laborious procedures that result in a rapid reduction of the volume of the cell culture supernatant to be processed without the need for a centrifugation step. one problem with the centrifugation and filtration-based methods outlined above is that cell-derived components are concentrated along with the vector particles. these have the potential to cause immune and inflammatory responses. for example, concentrated vsv-g-pseudotyped lv vector preparations were shown to be contaminated with tubovesicular structures of cellular origin which carried nucleic acids, including the plasmid dnas that were used to generate the lv vector stocks. dna carried by these tubovesicular structures acted as a stimulus for innate antiviral responses, triggering toll-like receptor and inducing alpha/beta interferon production. thus, additional steps including chromatography-based methods such as anion exchange chromatography are needed in order to reduce host cell and cell culture-derived contaminants from lv vector preparations. methods based on anion exchange column chromatography of lv vectors pseudotyped with vsv-g or the baculovirus gp glycoprotein were previously described. however, the yields and purity of the lv vector stocks obtained in this way were not reported. in an attempt to simplify the production and concentration of lv vectors and to make this approach more reproducible and cost-effective for preclinical studies in animals, we have worked out a facile lv vector production system based on hyperflasks. we also implemented a straightforward concentration procedure based on mustang q anion exchange membrane chromatography. mustang q anion exchange-based chromatography protocols for concentrating/purifying lv vectors were previously reported. such vector preparations displayed reduced toxicity compared to vectors concentrated using ultracentrifugation, as well as enhanced purity.  simplified production of lentiviral vectors using hyperflasks with a view toward improving high-titer lv vector production for preclinical studies in animals, we tested the usefulness of hyperflask vessels that have a total growth area of cm corresponding to ten standard t flasks. hyperflasks consist of ten interconnected growth surfaces each containing a membrane pretreated to allow improved cell adherence. the membrane is gas permeable, allowing exchange of oxygen and carbon dioxide through the base of the membrane, resulting in gas exposure to a large surface area within the flask. to test the usefulness of hyperflasks for lv vector production involving calcium phosphate-mediated transfection, we compared the titers of lv vector stocks prepared side-by-side using either ten 150-cm dishes or a single hyperflask. the data shown in table represent the results of three independent productions each for the 150-cm dish system and the hyperflask system. these data indicate that the titers of unconcentrated lv vectors produced using hyperflasks were up to × tu/ml while the titers of lv vectors produced in 150-cm dishes were lower, up to × tu/ml. the total yields were up to × tu from ten 150-cm dishes and up to × tu for the hyperflask vessels. this corresponds to a productivity of up to × tu per cm for the 150-cm dishes. for the hyperflask, the productivity was about 10-fold higher, up to × tu per cm the higher productivity observed with hyperflasks may be related to better gas exchange during lv vector production. overall, the 293t cells used for production appeared healthier and there was less cell debris from transfections carried out in hyperflaks compared to transfections carried out in 150-cm dishes. we expect the hyperflask production protocol described here to be compatible with other dna transfection formats such as polyethylenimine-mediated transfection. ten 150-cm dishes and one hyperflask vessel were used for lv vector production. productions were performed side-by-side. vector titers were determined using hos cells. data are expressed as the mean ± standard deviation. simplified concentration of lentiviral vectors using mustang q acrodiscs the concentration and purification of lv vector stocks on a large scale presents a significant bottleneck and methods allowing rapid processing of large volumes of lv vector-containing supernatants are needed. chromatography-based methods appear to be particularly attractive in this regard. for example, methods to concentrate/purify lv vectors based on anion exchange or affinity chromatography have been established. recently, segura et al. described an approach involving heparin affinity chromatography to concentrate lv vector particles directly from clarified supernatants. during this step, a recovery of 53% of infectious lv particles was achieved while 94% of the impurities were removed. this strategy may ultimately result in vector stocks of improved purity, increased infectivity and reduced toxicity. however, these approaches are cumbersome and difficult to implement in a standard laboratory setting. we have recently described a facile membrane-based chromatography approach involving mustang q anion exchange chromatography cartridges to purify adenoviral vectors. mustang q membranes resulted in high recoveries of infectious viral particles and allowed the processing of adenoviral vector particles from lysates in a fraction of the time required using the traditional cesium chloride-based ultracentrifugation method. mustang q membranes include a matrix that has a high dynamic binding capacity for adenoviral vectors and is capable of withstanding high flow rates. other benefits of mustang q membrane chromatography cartridges include higher peak resolution at faster flow rates as compared to traditional ion exchange resins. also, they can easily be adapted to bench-scale work and they are syringe-adaptable. finally, there is no need for an hplc setup or a column packing step. in work with lv vectors carried out by us and by others, mustang q-based cartridges were used to concentrate/purify lv vectors. while these initial studies were encouraging, the capacity of mustang q membranes for crude lv vector supernatants was rather low, possibly caused by cell-derived proteins, serum proteins, or contaminating nucleic acids that bound to mustang q membranes during vector capturing. to determine the binding capacity of mustang q membranes for lv vectors produced in hyperflask vessels compared to 150-cm dishes, a capture study was conducted. figure shows a typical elution profile of hyperflask-produced lv vectors following capturing onto a -ml mustang q acrodisc and elution using a gradient ranging from to m nacl. tu were determined by facs analysis of transduced hos cells. the results presented in table show that the capacity of mustang q acrodiscs at 10% breakthrough for lv vector supernatants produced in 150-cm dishes was up to × tu per ml of membrane while the capacity of mustang q membranes at 10% breakthrough for hyperflask vessel-derived lv vector samples was up to × tu per ml of membrane. this is consistent with the observation that lv vector supernatants produced using hyperflasks contained fewer cellular proteins and nucleic acids compared to supernatants produced in 150-cm dishes that may have interfered with the binding of the lv vector particles. to remove excess nacl, pooled mustang q fractions were subjected to a buffer exchange using sepharose cl-4b. table summarizes the recovery of lv vectors following processing of 500-ml aliquots of hyperflask vessel-derived supernatants. up to × tu were obtained after mustang q chromatography and up to × tu after subsequent buffer exchange using sepharose cl-4b. mustang q acrodiscs were challenged with 75-ml aliquots of vector-containing supernatants. the crude vector supernatants were filtered using a μm filter prior to anion exchange chromatography on mustang q acrodiscs. input corresponds to the total number of tu loaded. unbound corresponds the total number of tu found in the flow-through. bound represents input tu minus unbound tu. recovered corresponds to the total number of tu following elution. capacity corresponds to the total number of tu bound per ml of the mustang q membrane. since the hyperflask-derived samples did not result in greater than 10% breakthrough, the upper limit of the capacity for these samples is not known. titers were determined by facs using hos cells. data from three independent experiments involving 150-cm dish-derived and hyperflask vessel-derived vector supernatants are shown. the data are expressed as mean ± sd. protein and dna concentrations of 150-cm dish-derived and hyperflask vessel-derived vector supernatants were determined using a qubit assay kit. productions were performed side-by-side. data are expressed as mean ± sd. mustang q acrodiscs were challenged with 500-ml aliquots of hyperflask vessel-derived vector supernatants. buffer exchange was carried out by sepharose cl-4b size exclusion chromatography as outlined under materials and methods. titers were determined by facs using hos cells. data from three independent productions are shown. data are expressed as mean ± sd. finally, we wanted to compare the titers and total yields of our concentrated lv vectors with those reported by other groups. to do this, we carried out a side-by-side titer comparison of a hyperflask-derived lv vector stock concentrated by mustang q anion exchange chromatography using hos cells, 293t cells and hela cells. table shows that the titers of fresh vector stocks were similar in hos cells and 293t cells and slightly lower in hela cells. upon freezing and thawing of mustang q-concentrated vector stocks there was a 40% drop in vector titers on average. freshly concentrated lv vector stocks were diluted and aliquots were used to transduce hos, 293t and hela cells as outlined under materials and methods. titers were determined by facs. data are expressed as mean ± sd. overall, the titers obtained using the hyperflask system were as high or higher compared to those obtained using 150-cm dishes and ultracentrifugation-based concentration approaches. the yield from a single hyperflask was above tu. this is about two orders of magnitude higher than the yields reported by karolewski et al. who used a cell factory system, and at least one order of magnitude higher than the yields reported by geraerts et al. who also used cell factories.  in conclusion, we present here a simple protocol for lv vector production and concentration involving hyperflasks in conjunction with mustang q anion exchange membrane cartridges. these protocols are easy to implement in a standard laboratory setting and allow high-titer lv vector production without the need for multiple tissue culture dishes or flasks or ultracentrifugation-based concentration procedures. 
here we present a simplified protocol for lv vector production on a laboratory scale using hyperflask vessels. the protocol described here is easy to implement and should facilitate high-titer lv vector production for preclinical studies in animal models without the need for multiple tissue culture dishes and ultracentrifugation-based concentration protocols. hyperflask vessels are high-yield, high-performance flasks that utilize a multilayered gas permeable growth surface for efficient gas exchange, allowing convenient production of high-titer lv vectors. during the past twelve years, lentiviral vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells in vitro and in vivo. we also show that up to ml of an unconcentrated lv vector stock prepared using a hyperflask vessel could be concentrated using a single mustang q acrodisc with a membrane volume of ml. up to × tu were recovered from a single hyperflask vessel. our results show that unconcentrated lv vector stocks with titers in excess of transduction units per ml were obtained using hyperflasks and that these titers were higher than those produced in parallel using regular 150-cm tissue culture dishes. for subsequent concentration of lv vector stocks produced in this way, we describe a facile protocol involving mustang q anion exchange membrane chromatography. however, despite significant progress, the production and concentration of high-titer, high-quality lv vector stocks is still cumbersome and costly.
 during the past twelve years, lentiviral vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells in vitro and in vivo. however, despite significant progress, the production and concentration of high-titer, high-quality lv vector stocks is still cumbersome and costly. methods here we present a simplified protocol for lv vector production on a laboratory scale using hyperflask vessels. hyperflask vessels are high-yield, high-performance flasks that utilize a multilayered gas permeable growth surface for efficient gas exchange, allowing convenient production of high-titer lv vectors. for subsequent concentration of lv vector stocks produced in this way, we describe a facile protocol involving mustang q anion exchange membrane chromatography.  our results show that unconcentrated lv vector stocks with titers in excess of transduction units per ml were obtained using hyperflasks and that these titers were higher than those produced in parallel using regular 150-cm tissue culture dishes. we also show that up to ml of an unconcentrated lv vector stock prepared using a hyperflask vessel could be concentrated using a single mustang q acrodisc with a membrane volume of ml. up to × tu were recovered from a single hyperflask vessel.  the protocol described here is easy to implement and should facilitate high-titer lv vector production for preclinical studies in animal models without the need for multiple tissue culture dishes and ultracentrifugation-based concentration protocols. 