Purification virus protocol

Try out PMC Labs and tell us what you think. Learn More. Virus production is an essential part of virology. The quality of virus preparation, in terms of purification and yield, can affect the outcome of experiments.

Purified virus is critical for assays, as contamination from cell culture supernatants may affect results. Purified virus is also needed for vaccine preparations. The traditional process of gradient purification of virus is time consuming, with multiple steps spanning over days, and produces significant amounts of biohazard waste.

Developing a more efficient alternative that provides a purified virus product is highly desirable. Currently, this kit is only indicated for use with lentiviruses and retroviruses.

Therefore, we concluded that the Mag4C LV kit provides a quick and simple alternative to traditional virus purification methods. Virus production is an essential part of virology experiments and the quality of virus preparation in terms of purification and yield can affect the results of these experiments.

Purified virus is critical for assays where contamination from the cell culture, e. Purified virus is also needed for vaccine preparations [2]. The traditional process of gradient purification is time consuming, as it requires multiple steps Fig. Other methods for purification, such as size exclusion filtration, can still carry contaminants from the cell culture [3].

Thus, a more efficient method of effective virus purification is highly desirable. Process stages and length of time needed for sucrose gradient, size exclusion filtration, and magnetic bead purification.

After supernatant containing virus is collected from infected multi-layered flasks, it is centrifuged and sterile filtered, producing clarified supernatant. This clarified supernatant is further purified and concentrated by sucrose gradient, size exclusion filtration, or magnetic bead purification. Sucrose gradient : Clarified supernatant is ultracentrifuged and virus is pelleted. After the pellet is dissolved overnight in tris-EDTA-saline TES buffer, a sucrose gradient ultracentrifugation is performed and the bands containing purified virus are collected.

These bands are further concentrated with a final ultracentrifugation, and the resulting pellet is dissolved overnight in TES buffer. The total amount of time the filtration takes depends on the volume of clarified supernatant to be used. Magnetic bead purification : Magnetic beads are suspended in clarified supernatant and allowed to bind to the virus.

The bead-virus complex is separated from the supernatant, and the virus is eluted from the beads and collected. Magnetic beads can be used for many biological applications, such as immunoprecipitation [4] and multiplexing [5]. They can also be utilized for virus capture and concentration [6]. However, commercial magnetic bead purification kits are mainly indicated for lentiviruses, retroviruses, and adenoviruses.

In this study, we evaluated the efficacy of a commercial magnetic bead kit created for the concentration of lentiviruses and retroviruses, for use with an alphavirus i. Mayaro virus MAYV or a flavivirus i. Zika virus ZIKV. Vacuum filter 0. At this point, the beads should be held on the wall of the tube.

BCA results show that overall protein concentration decreases as more purification steps are performed Table 1 ; this is likely due to the protein content from the lysed cells or protein present in the cell culture medium being removed with each step of the purification protocol. Protein concentrations in each sample were measured via BCA assay. As more purification steps are performed, the protein concentration decreases.

This demonstrates how the magnetic beads are able to separate virus particles from other proteins in the clarified supernatant. The qualitative detection of total protein quantities i. The protein gel results correspond to the BCA results; specifically, as more purification steps are performed, less excess protein is observed on the gel, while the virus-specific proteins remain. It is also demonstrated that there are negligible amounts of protein remaining on the beads at the end of the magnetic bead purification process, indicating that the majority of virus has successfully been eluted.

The intensity of the band decreases as more purification steps are performed, indicating possible loss of virus, which is consistent with the titer data Table 2.

Similarly, MAYV samples showed binding to mouse anti-MAYV hyperimmune ascitic fluid with a decrease in band intensity as further purification is performed. The samples were serially diluted from 10E-1 to 10E and used to inoculate Vero cells in well plates. Each dilution had 8 replicates.

The virus titer decreases as more purification steps are performed by about 10 —1 , indicating loss of virus particles during the purification process. Characterization assays provided insight into the quantity of virus is in each sample as well as the purity of each sample. We also compared the quantity and purification with the time needed to generate each sample via different methods.

Although sucrose gradient purification is the standard for purification in the field, it is time-consuming, taking 2—4 days to complete. Additionally, it is difficult to produce a highly concentrated, pure sample and also requires specific instrumentation including a gradient maker and fractionator.

Size exclusion filtration can be performed quickly, but the quality of purification is very poor. The developed magnetic bead purification protocol can be used for the purification of ZIKV or MAYV as well as other viruses in the respective groups with minimum instrumentation need yet produces purified virus.

However, the loss of virus with each purification step may limit the use of this approach where very high concentration and yield may be needed in downstream applications. The eluted virus may then be further concentrated by either centrifugal spin devices or tangential flow filtration yielding material of high titer and Good Manufacturing Practice GMP grade biochemical purity.

The protocol is validated for rAAV serotypes 2, 8, and 9. The described method makes rAAV vector technology readily available for the low budget research laboratories and could be easily adapted for a large scale GMP production format. Recombinant adeno-associated virus rAAV vectors have emerged as one of the most versatile and successful gene therapy delivery vehicles.

A number of recent clinical trials had impressive clinical outcomes 1 , 2 , 3 , 4 , 5 , 6 and patients diagnosed with lipoprotein lipase deficiency will now have an option to be treated with Glybera, the first rAAV-based drug to win the regulatory approval of the European Medicines Agency.

However, even though the industry is poised for the expansion into several application areas represented by orphan diseases, a simple and scalable rAAV production technology is still lacking. The ever growing rAAV vector toolbox, in addition to many natural AAV serotypes, now includes numerous AAV capsid mutants derived from combinatorial libraries or through rational engineering. Although quite useful in a laboratory setting, these procedures are neither scalable nor easily adapted for Good Manufacturing Practice GMP protocols.

In this regard, the more promising approach incorporates chromatography steps, either affinity, hydrophobic, or ion-exchange, depending on the biochemical properties of a particular serotype. For example, heparin affinity chromatography based on interaction with heparan sulfate proteoglycan has been successfully applied to rAAV2, 8 , 9 while mucin affinity chromatography can be used for rAAV5 purification because it binds to sialic acid.

In this report, we describe an efficient and reproducible protocol based on a partial purification of the initial crude lysate by flocculation of cell debris under low pH conditions, followed by one-step cation-exchange chromatography. The flocculation step eliminates the bulk of the contaminating protein and DNA allowing for quantitative AAV binding to, and subsequent elution from the resin.

The method could be applied to several serotypes and for vectors purified from both mammalian and insect cell production systems. AAV9 was selected for the development of all the experimental steps since this is one of the most challenging AAV serotypes to purify. A physiological solution, Lactated Ringer pH6. As shown in Figure 1a , after a short 2-hour exposure, the infectivity of the virus does not change in any of the buffers over the whole pH range tested.

After hour incubation, however, there was a tenfold reduction in infectivity which was more pronounced at the range of pH, followed by another log reduction after exposure for 3 weeks. Surprisingly, however, the lower pH range was less deleterious thus providing experimental validation for the low pH-induced flocculation step.

Obviously, other components of the buffer provide structural stability as well because Lactated Ringer appears to better sustain higher virus infectivity over 3 weeks, the period of time tested.

After incubation, an aliquot was diluted in Lactate Ringer solution and used to infect C12 cells coinfected with Ad5 multiplicity of infection of 5. Table 1 lists the last temperature at which intact capsids were detected HL and the first temperature at which denatured capsids were detected B1 and A1.

At neutral pH pH 7. Low pH pH 4. However, intermediate acidity pH 5. Interestingly, there appears to be a reverse correlation between increased capsid stability in the pH 5. As with the dot-blot assay, capsids in citrate-phosphate buffer at pH 7. However, above these respective temperatures the capsids were denatured and no longer visible.

In addition, for capsids at pH 6. These EM results agree with the dot-blot data and together these observations demonstrate a very sharp transition temperature in capsid stability, which is otherwise very high for the rAAV9-GFP capsids.

AAV9-based vectors exhibit distinctive properties such as delayed blood clearance, ability to cross blood—brain barrier, and targeting cardiac muscle with a higher tropism. This serotype, however, proved to be challenging to purify using standard chromatography procedures. Followed flocculation step, we tested whether AAV9 remaining in the supernatant was capable of binding to an ion-exchange resin Figure 2a.

Thus, low pH flocculation followed by low-speed centrifugation disposes of The additional faint bands in the purified sample seen on the silver-stained gel are VP-derived peptides Figure 2c , right panel. EM examination of rAAV9 capsid purified by SP column chromatography Figure 2d reveals a higher degree of purity and integrity of the sample as compared to the one purified by iodixanol gradient Figure 2e. Summary of rAAV9 purification. Negative stain images of rAAV9 purified d using the sodium citrate method, or e using discontinuous step gradient of iodixanol.

Filled arrowheads point at the DNA-containing particles; empty arrowheads: at the empty capsids; black asterisk indicates proteasome; white asterisk indicates deformed AAV particle. The missing bars for some steps indicate the titers below 10E4, the Y-axis plotting scale.

AAV2 is one of the most utilized and studied serotypes providing structural platform for rationally designed vectors and combinatorial capsid libraries. Summary of rAAV2 purification. AAV8 serotype-based vectors are among the most efficient vehicles for transducing target tissues in vivo , and they are widely utilized both in the laboratory setting and in clinical trials.

Moreover, several laboratories have designed insect cell-based systems to scale up production of rAAV8 vectors. If this peptide is a product of capsid autocleavage, then perhaps it happens at a somewhat higher rate under the low pH conditions used in this protocol.

Summary of rAAV8 purification. The purpose of the current project was to develop a simple and reproducible purification method applicable to many, if not all, AAV serotypes and variants.

In addition, the method should be applicable to AAV vectors produced from either mammalian or Sf9 insect cell cultures, i. Previously, we conducted an in-depth study of methods for the recovery of rAAV from bulk cell lysates establishing that no satisfactory method existed among those tested at the time. Moreover, sodium citrate buffer was selected for the subsequent chromatography step because of its working pH range of 3. Thus, the flocculation and subsequent binding to SP resin could be carried at pH3.

Prior to the method development we investigated whether exposing virus to low pH modifies its structure rendering it noninfectious. Consistent with the previous observations, 18 acidification of the virus for 2 hours resulted in little if any reduction of the infectivity. Although longer incubations were somewhat unfavorable, the actual time of the purification flocculation followed by column chromatography is compatible with short low pH exposure times.

In this report, we describe a simplified purification protocol which, regardless of the upstream production method, yields exceptionally pure vector preparations. To our knowledge, this is one of the most inexpensive protocols utilizing simple off-the-shelf reagents such as sodium citrate and citric acid. Separating DNA-containing and empty rAAV particles without ultracentrifugation in buoyant density gradients remains a technical challenge. Curiously, the pI value for virions incorporating packaged DNA is different from those for empty capsids.

In summary, we have developed an affordable protocol for the purification of rAAV using off-the-shelf reagents and easy to follow steps. Because of its overall simplicity, the protocol could be used in a regular research laboratory, as well as further adapted for GMP-grade industrial scale production. Cells were harvested 72 hours later and rAAV was purified as described below. To maintain the reproducibility regardless of the scale of the process the volumes of each of the reagents used in this protocol are based on the approximate wet weight of the harvested cell pellet.

After low-speed centrifugation, the supernatant is subjected to ion exchange chromatography. Schematic flowchart of adeno-associated virus AAV purification protocol. Step 1: A crude cell lysate is acidified by the addition of citric acid. Step 2: A heavy flocculate is precipitated by a low spin centrifugation. Step 3: A supernatant is subjected to a one-step cation-exchange chromatography. Heavy flocculate, which immediately formed at this point, was precipitated by centrifugation at 4, g for 10 minutes at room temperature.

Forty-eight hours later, cells infected with rAAV-GFP were visually scored using a fluorescence microscope and the titer was calculated according to the dilution factor.