Frequently Asked Questions on Transfection Reagents and pDNA Products

FectoVIR^®-AAV is a transfection reagent used as raw material in AAV production process. FectoVIR^®-AAV gives higher productivity than other commercially available transfection reagents and decreases global cost of production.

The FectoVIR^®-AAV structure is confidential and a patent has been filled by Polyplus. PEIpro^®, FectoVIR^®-AAV and FectoVIR^®-LV are different both in terms of molecular structure and formulation.

Expiry date is indicated in the Certificate of Analysis available in ”My Sartorius”.

We have successfully used FectoVIR^®-AAV on suspension HEK-293 derivatives: HEK-293T, HEK-293F, Expi293, VPC, VPC2.0, etc... Customers confirmed that FectoVIR^®-AAV works well in several commercial and proprietary suspension HEK-293 subclones.

In house, we produce both AAV-2, AAV-5, AAV-8 and AAV-9. More serotypes have been tested by our customers, with comparable yield improvements. 

FectoVIR^®-AAV shows a remarquable improvement in terms of viral titers in suspension and adherent systems.

For lentivirus production, we recommend FectoVIR^®-LV as it is currently the best product for this application, both in adherent and suspension cell culture systems.

FectoVIR^®-AAV GMP is manufactured in compliance with the ICH Q7 GMP part II guidelines that ensure a validated and aseptic manufacturing process. In addition, FectoVIR^®-AAV GMP is provided with a full regulatory documentation to support IND and ensure the full traceability of the product. Using pharmaceutical guideline to manufacture a transfection reagent will help you reduce the risk and ensure patient safety. 

FectoVIR^®-LV is the next generation of transfection reagent to produce lentiviral vectors used for gene modified cell therapies (CAR-T, CAR-NK, gene modified stem cell therapy etc..). It tooks 3 years to develop this new molecule and finally found a transfection solution that enables to increase LVV titers.

Expiry date is indicated in the Certificate of Analysis available in ”My Sartorius”.

FectoVIR^®-LV GMP is currently under development.

PEIpro^® has been specifically developed for large-scale virus production (mostly AAV and LV but other viruses as well) in both adherent and suspension cells grown either in serum-containing or serum-free medium. PEIpro^® is provided with extensive QCs making it suitable for therapeutic virus production.

PEIpro^®, PEIpro^®-HQ and PEIpro^®-GMP are composed of a linear polyethylenimine (PEI) whose molecular weight is proprietary information. Extensive optimization experiments have been performed to select a linear PEI for optimal DNA delivery with the lowest cytotoxicity in vitro.

PEIpro^® is provided as an aqueous solution of linear PEI. 

PEIpro^® can undergo an accidental freeze/thaw cycle without compromising the transfection efficiency. However, for long term storage we recommend storing the reagent at 5°C ± 3°C to ensure long term stability.

Expiry date is indicated in the Certificate of Analysis available in ”My Sartorius”.

Depending on the type of viral vector, different subtypes of HEK-293 cells are preferred such as HEK-293 for adenovirus and AAV virus production, HEK-293T and HEK-293FT for AAV and lentivirus production. PEIpro^® is suitable for virus production in both adherent and suspension subtypes of HEK-293 cells.

Different culture vessels including multilayers flasks such as HYPERFlasks™ and HYPERstacks™ (Corning), or fixed-bed culture systems such as iCELLis^® bioreactors (Pall) are compatible with the use of PEIpro^® in adherent HEK-293 cells. For virus production in suspension HEK-293 cells, large size shaker flasks or bioreactors can be used.

In contrast to many other transfection reagents, all our products are compatible with serum therefore complexes can be directly added to cells in serum-containing medium. 

None of our products transfection efficiency is affected by the presence of antibiotics. For example, the routine validation protocol for each new batch of jetPRIME^® is always performed in serum and antibiotics containing medium. 

Many cell culture media are used to grow and maintain mammalian cells and some of them may inhibit cationic polymer/lipid-mediated transfection. For more information about cell medium compatibility with transfection, contact our delivery experts.

Passage numbers will affect transfection performance. If cells have been in culture for a long time (over 20 passages), transfection efficiency will decrease and we recommend starting a new culture with a fresh tube of cells from liquid nitrogen. On the other hand, cells should be passaged at least twice after thawing before starting transfection experiments.

Cell density will affect transfection performance. The recommended confluence at the time of transfection can vary according to the transfection reagent. Please refer to the reagent protocol for the recommended number of cells to seed to obtain the expected confluence on the day of transfection 

The ratio corresponds to the nucleic acid amount, in µg, per microliter of transfection reagent to use For example, a 1:2 ratio means 1µg of nucleic acid per 2 µl of transfection reagent. 

There are several parameters that can be changed to improve the transfection efficiency. If you are looking for some optimization, contact our Scientific Support team at support@polyplus-transfection.com. 

jetOPTIMUS^® is our recommended reagent for day-to-day transfection of adherent cells, even for hard-to-transfect cells. It is well suitable for co-delivery of several plasmids and small-scale virus production. Transfection with jetOPTIMUS^® leads to higher transfection efficiencies and gene expression with minimal impact on cell viability and morphology.

jetPRIME^® is a polyvalent transfection reagent that ensures effective and reproducible DNA and siRNA transfection in several adherent cell lines. This versatile reagent is also perfect for co-transfection of DNA and siRNA. Transfection with jetPRIME^® requires only low amounts of nucleic acid, hence resulting in very low cytotoxicity.

jetPEI^® transfection reagent is particularly well suited for automated or manual HTS (High Throughput Screening) with three protocols available: reverse, batch and forward. jetPEI^® provides highly reproducible results and the DNA/jetPEI^® complexes are stable for up to 4 hours.

For co-transfection of multiple plasmids, we recommend that the total DNA amount per well does not exceed the maximal DNA amount indicated in the jetOPTIMUS^® protocol. The ratio to use for each plasmid depends on the size of the plasmid, the plasmid construct and the expected expression level of each plasmid. Each plasmid should represent at least 10% of the total DNA amount per well.

For plasmid DNA/siRNA co-transfection, we recommend jetPRIME^® reagent. Please refer to the standard protocol for DNA and siRNA co-transfection available on the 

For DNA/ssODN co-transfection (single-stranded oligodeoxynucleotides), the same protocol may be applied.

jetOPTIMUS^® is well suitable for DNA transfection of hard-to transfect cells such as primary cells (epithelial, hepatocyte, endothelial, fibroblast, etc…), stem cells (embryonic, mesenchymal, induced pluripotent) and cancer cell lines.

However, some suspension and primary cells, such as lymphocyte cell lines, fibroblasts or neurons, are very difficult to transfect with DNA Polyplus^® developed a lipid-based transfection reagent jetMESSENGER^®. It was designed for mRNA delivery and showed high efficiency on a wide variety of difficult to transfect cells including lymphoma and blood cell lines, neurons and various primary cells. Compared to DNA, mRNA transfection leads to a high percentage of transfected cells, faster protein expression following transfection, and no risk of genomic insertion in contrast to plasmid or viral vectors

For optimal transfection efficiency using jetOPTIMUS^® or  jetPRIME^®, we recommend not exceeding 10 minutes of complexation. Hence, for applications requiring longer incubation times such as High Throughput Screening, we recommend using jetPEI^®, as jetPEI^®/DNA and DNA/jetOPTIMUS^® complexes are stable for up to 4 hours.

There is no size restriction for DNA transfection using those reagents, However, it should be pointed out that for the same amount of DNA, the gene copy number will be lower for large DNA constructs than for smaller plasmids. 

jetOPTIMUS^® interacts with DNA, forming positively charged complexes which penetrate into the cell through endocytosis. Endosomes release DNA in the cytosol via the proton sponge mechanism (Behr, Chimia 1997). The plasmid mostly reaches the nucleus during mitosis, when nuclear envelope disappears. For this reason, primary cells or cell lines with slow cell division rates are usually hard-to-transfect.

INTERFERin^® is the reagent of choice for siRNA transfection, and works with as little as 1 nM siRNA.

jetPRIME^® is suited for siRNA delivery at higher concentrations (using 10 to 50 nM) and is recommended in case of DNA/siRNA co-delivery.

Furthermore, if you want to deliver siRNA or miRNA in vivo, our best suited reagent for this application is in vivo-jetPEI^®.

shRNA can be transfected via a plasmid vector or as oligonucleotides. If shRNA are transfected as oligonucleotides, then INTERFERin^® is recommended. However, if a vector expressing the shRNA is used then jetOPTIMUS^® jetPRIME is a more suitable reagent. For in vivo delivery of shRNA, we recommend using in vivo-jetPEI^®.

Our in vivo–jetPEI^® is a powerful polymer-based reagent, suited for delivery of oligonucleotides to any animal model. The protocol is similar to a classical transfection: the nucleic acid and the reagent are just mixed and administered to the animal. 

We recommend using jetPRIME^® reagent for delivery of single-stranded oligonucleotides.

For plasmid DNA and ssODN co-transfection (e.g. CRISPR/Cas9 applications), please refer to the standard protocol we provide for DNA and siRNA co-transfection.

Several types of molecules are abusively called miRNA. The term miRNA can refer to:

mimic miRNA: these are structurally similar but not identical to siRNA and can be transfected with INTERFERin^®.
plasmid encoding for miRNA: this molecule can be transfected using jetOPTIMUS^®.
antimiR: this is a single-stranded nucleic acid designed to specifically bind to and inhibit endogenous microRNA; it can be transfected using INTERFERin^®.

For the same amount of transfected oligonucleotide, the number of delivered copies will be different depending on the oligonucleotide size (longer oligonucleotide: less copies, shorter oligonucleotide: more copies). Therefore, oligonucleotide amounts should be adjusted accordingly. 

For in vitro mRNA transfection, jetMESSENGER^® is our reagent of choice. Indeed, jetMESSENGER^® protocol is simple: the reagent is ready-to-use and is compatible with the standard growth media (with or without serum and antibiotics) of a wide variety of adherent and suspension cell types. jetMESSENGER^® operates through a very gentle process and upholds cell viability and overall cellular morphology in a variety of hard to transfect cells, such as rat cortex neurons, human liver carcinoma cells and mouse embryonic stem cells.

For in vivo mRNA delivery, in vivo-jetRNA^®+ is our best-in-class reagent. in vivo-jetRNA^®+ is a ready-to-use transfection reagent composed of preformed liposomes specifically developed to deliver mRNA in vivo. This reagent can be used to target unique or multiple organs, by using systemic injection routes, in various animal models (mice, rat, etc.). mRNA delivery using in vivo-jetRNA^®+ is user-friendly, with a simple 2-step protocol and is highly efficient with comparable delivery results as Lipid Nanoparticles (LNPs). in vivo-jetRNA®+ can be used for vaccination/immunization purposes, anti-cancer studies, genome editing using CRISPR/Cas9 method or protein replacement.

With the LipidBrick^® family, we also offer a range of cationic lipids dedicated to the formulation of lipid nanoparticles (LNPs). These active lipids protect the mRNA molecules and play an important role in the transfection capacity of LNPs. Importantly, by being based on an imidazolium polar head, LipidBrick^® broadens the spectrum of current LNP applications in terms of potency and biodistribution by adding an overall positive charge to LNPs: this translates into greater delivery of mRNA to the lungs and spleen while reducing accumulation in the liver compared to LNPs based on ionisable lipids. Furthermore, within the LipidBrick® family, LipidBrick^® IM21.7c is the cationic lipid (active lipid) used in the formulation of jetMESSENGER^® and in vivo-jetRNA®+, allowing a seamless transition between our ready-to-use reagents and your LNP formulation tailored to your needs and applications.

mRNA can be in vitro transcribed (IVT) from DNA template using commercially available solutions (e.g. HiScribe™ kit provided by New England Biolabs) or be purchased “custom made” through different oligonucleotide suppliers such as Trilink, Eurofins, Aldevron, etc.

With our D-zyvec platform, we can design and linearized DNA templates specifically tailored to your mRNA needs. Please feel to contact our Scientific Support team for more information (contact us).

Of note, DNA template template must be linearized and contain a RNA polymerase promoter region such as T7 or SP6 upstream of the sequence to be transcribed, in 5’ for the sense RNA or in 3’ for the antisense RNA. If required, the RNA polymerase promoter region can be added upstream of the sequence to be transcribed by PCR using primers containing the minimal promoter sequences.

Generally speaking, mRNA purification following in vitro transcription is not necessary, as there should be minimal RNA contaminants. If needed, several methods are suitable, including Phenol-chloroform Extraction/Ethanol Precipitation, LiCl precipitation, gel purification methods, and several commercially available mRNA isolation kits.

There is no limit in mRNA size per se. It is worth to keep in mind that mRNA synthesis kits are usually optimized for mRNA transcripts ranging in size from 0.3 to 5kb, equivalent to a protein size of 10-150 kDa. Therefore, for shorter or longer mRNA transcripts, additional optimization may be required to obtain a similar high yield.

In comparison to DNA transfection, mRNA-mediated transfection with jetMESSENGER^® and in vivo–jetRNA^®+ enables a more controlled gene expression that is not promoter dependent, over a similar period of time. In actively dividing cells, mRNA expression will be more prominently and stably observed between 1 to 4 days post-transfection. In slow to non-dividing cells, mRNA expression can be stable over a longer period of time, as for example up to 7 days in primary neuronal cells.

During the synthesis of endogenous eukaryotic mRNA, a 7-methylguanosine CAP is added in 5’ on the nascent transcript to protect it from degradation and increase translation rate. The presence of a polyA-tail in 3’ of nascent mRNA further protects from degradation and plays a role in the nuclear export of the mature mRNA. Therefore, existing mRNA synthesis kits usually offer solutions to improve stability of mRNAs by incorporation of 5’ cap analog, and 3’ polyA-tail. In addition, specific base modifications within the mRNA sequence such as 5-methylcytosine and/or N6-methyladenine can be incorporated for increasing stability and decreasing immunogenicity. Custom-made mRNA ordered from an oligonucleotide provider can be ordered with specific modifications such as Pseudo-Uridine for increased stability.

Increasing the stability of the RNA transcript may not always be necessary. For example, some viral RNA transcripts do not need to be capped and polyadenylated in order to start replicating and/or produce infectious virus after transfection.

mRNA delivery is suited for transient expression, but not for the generation of stable clones. mRNA cannot integrate into the cell genome, which prohibits the generation of stable clones but has the advantage of not modifying the genome of the host cell. 

CRISPR mediated gene editing requires the delivery of the guide RNA and Cas9 protein into target cells using transfection. The validated method is based on the delivery of guide RNA and Cas9 protein as plasmid DNA, for which we recommend our latest technology jetOPTIMUS^®. jetOPTIMUS^® ensures highest gene editing efficiency in the largest range of primary cells and cell lines.

DNA-free gene editing methods are considered when off-target effects using the traditional plasmid-based method is a concern. Delivery of gRNA and Cas9 protein as mRNA is the most frequently used DNA-free alternative to reach robust gene editing efficiency in both adherent and suspension cells.

For a full RNA approach – mRNA encoding Cas9 and guide RNAs – we recommend our jetMESSENGER® transfection reagent.

Lastly, for a mix approach – plasmid DNA encoding Cas9 and gRNA as RNA- the most adapted reagent would be jetPRIME^®.

For in vivo CRISPR mediated gene editing, our reagent of choice is in vivo-jetPEI^®. Several groups have published their results using in vivo-jetPEI^® for gene editing in mice:

Zuckermann, M. & al. (2015). Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling. Nat Commun 6, 7391 

He, S. & al.  (2016) Annexin A2 Modulates ROS and Impacts Inflammatory Response via IL-17 Signaling in Polymicrobial Sepsis Mice. PLoS Pathog 12, e1005743 

Takao T. et al. (2020) Optogenetic regulation of embryo implantation in mice using photoactivatable CRISPR-Cas9. Proc Natl Acad Sci U S A 117 28579-28581 

Pallarès-Masmitjà M. et al. (2021) Find and cut-and-transfer (FiCAT) mammalian genome engineering. Nat Commun 12 7071 

McFadden T. et al. (2022) Short-term exposure to an obesogenic diet causes dynamic dysregulation of proteasome-mediated protein degradation in the hypothalamus of female rats. Nutr Neurosci 1-13 

in vivo-jetPEI^® is a ready-to-used polymer-based transfection reagent suitable for delivery of any type of nucleic acid such as DNA, siRNA, mimic miRNA and other oligonucleotides in vivo.

in vivo-jetRNA^®+ is a ready-to-used lipid-based transfection reagent which has been specifically developed to deliver mRNA in vivo.

LipidBrick^® is a range of cationic lipids dedicated to the formulation of lipid nanoparticles (LNPs). These active lipids protect the mRNA molecules and play an important role in the transfection capacity of LNPs.

in vivo-jetPEI^®  is ideal for nucleic acid delivery in mice. However, the protocol is so easy and versatile that it has been adapted to many other species including rats, mosquitoes, shrimps, guinea pigs, macaques…

Regarding in vivo-jetRNA^®+, it is also a ready-to-use reagent with a very simple 2-step protocol. Being a more recent reagent (launched at the end of 2022), it has so far been used in mice and rats.

Please feel to contact our Scientific Support team to adapt the protocol to your animal model contact us at polyplus-Support@Sartorius.com

in vivo–jetRNA^®+ and in vivo–jetPEI^® are suitable for systemic and local nucleic acid delivery. Several ways of administration have been successfully tested such as intravenous injection (tail vein and retroorbital), intraperitoneal injection, intratumoral injection, intracerebral injection, intranasal instillation.

The delivery procedure highly depends on the organ to target. Depending on the route of administration, in vivo-jetRNA^®+ or in vivo-jetPEI^®-mediated gene expression is observed in the brain, liver, pancreas, spleen, kidney, heart, bladder, skin, retina, etc.

Importantly, by being based on an imidazolium polar head, LipidBrick^® broadens the spectrum of current LNP applications in terms of potency and biodistribution by adding an overall positive charge to LNPs: this translates into greater delivery of mRNA to the lungs and spleen while reducing accumulation in the liver compared to LNPs based on ionisable lipids.

Please contact our Scientific Support team for more advice at polyplus-Support@Sartorius.com

Complexes formed between nucleic acid and in vivo-jetPEI^® do not form aggregates over time and are stable for at least 4 hours at room temperature or 37 °C and for up to 7 days at 4°C.

On the other hand, mRNA/in vivo-jetRNA^®+ complexes are stable for at up to 72 hours at room temperature and for up to 1 month at 4°C.

Hence, complexes can be prepared in advance, which is therefore compatible with the use of osmotic pumps for continuous delivery.

We offers a GMP grade in vivo-jetPEI^® which is manufactured in compliance with both US and EU current Good Manufacturing Practices (cGMP) suitable for use in clinical trials in Human from phase I to phase III. This reagent is used in a growing number of therapies worldwide.

Although in vivo-jetRNA^®+ has yet to be available in GMP grade, there is the possibility to develop GMP grade upon customer request (involving 12 to 18 months of development and a minimum order quantity).

FectoPRO^® is the reagent of choice for biomanufacturing of proteins in suspension as it has been developed for scientists interested in obtaining best possible results with mammalian Transient Gene Expression systems. FectoPRO^® ensures highest transfection efficiencies and protein yields in CHO and HEK-293 cell lines grown in variety of media. Moreover FectoPRO^® shows great cell viability, reproducibility and consistency in results. FectoPRO^® outperforms any other commercially available reagent and allows to optimize mammalian bioproduction processes (whatever scale used) as it requires equally less DNA and reagent.

For large or very-large scale experiments, you might want to consider PEIpro^® as it is also very efficient in HEK-293 cells and cost-effective.

In the case of protein production in an adherent system, PEIpro^® or jetOPTIMUS^® may be considered.

FectoPRO^® can be used without FectoPRO^® booster, but it is recommended if you want to achieve high protein yields using low DNA amounts.

FectoPRO^® works beautifully in regular and high cell density systems. FectoPRO^® allows to reach higher yields in a shorter timeframe – No need to wait for 7 or 12 days of cell growth to produce recombinant proteins.

The optimal DNA amount, when used with the medium is 0.8 µg DNA per mL of cell culture as stated in the FectoCHO^® Expression system protocol. If you use FectoPRO^® with any other media refer to the FectoPRO^® protocol for guidance. However, it should be noted that with the synergistic association of the FectoCHO™ CD Expression medium and the FectoPRO^® powerful transfection reagent you will obtain better protein production yields.

Both FectoPRO^® and PEIpro^® hasve been shown to work beautifully with HEK293 cells in suspension and serum-free media. But it depends on what your application is: FectoPRO^® is used for protein production whereas PEIpro^® is used for virus production.

No, you don’t need to buy the medium first and the transfection reagent in a second time since the FectoCHO™ Expression system kit is perfectly suited to both adapt your cells to FectoCHO™ CD Expression medium and perform transfection using FectoPRO^® transfection reagent. Indeed, in the FectoCHO™ Expression system kit (ref# 101000011), we provide you with 1L of medium and 1mL of transfection reagent, a perfect ratio of both reagents to allow you to conduct a full experiment from cell adaptation to protein production. Feel free to contact us for any pricing inquiry.

Depending on the medium used, 1 mL FectoPRO^® is sufficient to transfect between 500mL and 1L of culture.

The DNA amount per mL is the same if you use either the FectoCHO^® expression system or the ExpiCHO™ expression system in ExpiCHO™ cells. However keep in mind that, with FectoCHO^®, you can obtain up to twice the protein production yield at a lower cost.

FectoCHO^® CD medium can be used for culture and transient protein expression with ExpiCHO^® cells. However, it is important to note that it is not a plug and play system. A preliminary phase of adaptation and optimization is required.

Lipid nanoparticles (LNP) are the only clinically approved non-viral RNA delivery vehicle. Their safety has been demonstrated by the successes of mRNA vaccines after the recent coronavirus outbreak. COVID-19 vaccines rapidly adopted LNPs due to their ease of formulation, protective properties, and large payload capacity. LNPs are now accelerating the development of mRNA therapeutic applications in humans beyond vaccination.

Further developments are still needed to improve biodistribution and stability of mRNA-LNPs while reducing their toxicity. The ionizable cationic lipid within the LNP formulation influences which organs are transfected as well as mRNA delivery efficiency and degradability. Polyplus developed a range of cationic lipids used in LNP formulation named LipidBrick^®. These lipids can modulate physicochemical properties and impact subsequent potency and biodistribution of formulated LNPs while improving mRNA stability.

We deliver “discovery grade*” plasmids as circular, double stranded DNA molecules, in a screw-cap plastic tube, re-suspended in 1X AE buffer (miniprep.) or 1X TE buffer (midi, maxi, giga prep.), at room temperature. Please note that we DO NOT provide bacterial stock.

Discovery grade plasmids can be ordered at multiple formats, please inquire for more information.

For larger scale or higher quality grade plasmid production please inquire Plasmid Manufacturing Service

*Discovery grade means that while plasmids are produced in a sterile environment according to good laboratory practice, quality control are limited to basic quality and identity test. Discovery grade plasmids should be restricted to Research Application.

QC are limited to basic quality and identity tests:

• DNA quantification
• A260/280 and A260/230 measurements
• Restriction digest profile (gel or capillary electrophoresis)
• Whole plasmid sequencing including mycoplasma and gDNA detection (NGS)
• Sanger sequencing (if necessary)

QC results will be documented in the Product Delivery Form.

Please note that endotoxin levels ARE NOT measured for discovery grade plasmids.

For most projects, you do not need to send us existing plasmids or other DNA material. Desired sequences can be generated using DNA synthesis (outsourced).

However, some project may require you to send us DNA material (plasmid templates or purified gDNA, only) that we can use to overcome technical complexity, to ensure technical continuity or simply to reduce cost.

In this case, please follow these instructions:

• Use a 1.5 ml plastic tube, hermetically closed (screw cap or parafilm)
• Label the tube clearly
• Minimal volume: 15µL
• Minimal concentration: 100ng/µL
• Room temperature, please avoid dry ice.

Tubes should be sent at:

Sartorius Polyplus
Production Team
80 rue du Dr Yersin
59120 Loos
FRANCE

Please note that before starting any project, every plasmid template will be fully sequenced by NGS.

For gDNA samples please send a picture of an agarose gel to evaluate the integrity of the sample.

We do not perform DNA synthesis in-house, this is outsourced.

We have been successful in assembling plasmid up to 30Kb. Larger size plasmid can be considered as difficult to amplify in regular competent E. coli strains. However, feel free to reach out if your project requires such large plasmid as specific solutions may applied.

We operate two levels of services: STANDARD and PREMIUM,

• STANDARD Service allows working with customer material and can be delivered from 5 weeks*
• PREMIUM Service is solely based on DNA synthesis and can be delivered from 3 weeks​*.

* Upon receipt of the purchase order confirmation

Upon receipt, briefly spin down the tubes containing your engineered plasmids (bench centrifuge) and store them at:

•  4°C for short storage (<1 month)
• – 20°C for long storage

We strongly recommend to aliquot the Polyplus discovery grade plasmids for long storage and to avoid freeze-thaw cycles.

Duration of storage are given here as indication. You should always control the integrity of long stored plasmids by running gel electrophoresis (denaturation) or sequencing (sequence integrity).

Engineered discovery grade plasmids can be amplified by yourself for any Research Application.

Our bacterial origin of replication is functioning the same way as classical pBR322/pUc plasmids. You can therefore use any E. coli strain, accordingly to provider guidelines. We also indicate the culture temperature and the antibiotic selection we used to amplify your plasmids in the Product Delivery Form. As an indication, the concentrations of antibiotics we use routinely are 100µg/mL for ampicillin and 30µg/mL for kanamycin.

We do offer a pDNA amplification service for either engineered plasmids we generated, or your own plasmid you may wish to send us.

Amplification can be performed at small scale (up to several mg) and discovery grade, or at large scale (up to several gram) at R&D, High Quality or GMP grade with our Plasmid Manufacturing Service.

Unless otherwise stated in the specifications of you project, every engineered plasmid provided on a service based contract, for Research Application, are IP free from Polyplus, as stipulated in our T&C. Other applications may require licensing agreement with Polyplus or tiers entity.

Polyplus plasmid sequencing services are dedicated to whole plasmid sequencing. Therefore, starting material should be purified double strand DNA plasmids.

Please note that we do not perform whole genome sequencing of any organism.

You can send your samples following the guidelines below:

• Use a 1.5 ml plastic tube, hermetically closed (screw cap or parafilm)
• Label the tube clearly
• Minimal volume: 15µL
• Minimal concentration: 100ng/µL

Samples should be sent to:

Polyplus-Transfection
Production Team
80 rue du Dr Yersin
59120 Loos
FRANCE

When known, please enter the sequences of reference of your samples on our platform.

We operate two levels of services: Standard and Express.

• Standard Service offering comprehensive results from 2-3 weeks upon purchase and sample receipt. This format enables you to sequence even one single sample.
• Express Service guaranteeing the results within 3 working days upon purchase order and sample receipt. This format is quoted by batch of 12 samples, However, if you are in a hurry to get your results, you can request a batch of 12 samples to only few of them

After sequencing your sample, we will provide you the full-length sequence of your plasmid (.fasta file) as well as a sequencing report (.pdf file) containing:

• A visual representation of the sequencing results with the coverage and the differences observed with the sequence of reference, if there is any.
• The positions and the nature of the variations (insertion, deletion, substitution) observed in comparison with the sequence of reference.
• The percentage of  contaminations contained in your sample such as bacterial gDNA .

Sequencing report and the full-length sequence of your plasmids are available on your account.

We do perform de novo analysis.

We do NGS even for a single sample. There is no limitation in the number of samples, per se. We can sequence 150 samples at the same time.

Currently, we perform NGS using the Illumina technology (short reads, 150 bp).

We do offer consulting. If you need a specific bioinformatical analysis of your sequencing results, please contact us.

The online pDNA services platform has been tested on Mozilla Firefox, Google Chrome, and Microsoft Edge. While it functions on other browsers like Opera or Safari, complete compatibility across all features cannot be guaranteed.

We can view the projects you are currently working on, but we will not make any alterations or suggestions unless specifically requested.

There is no limitation of time between the creation and the submission of a project. For instance, you can save a project and return to it even months later to continue your work

When you submit a project, a plasmid specialist will get back to you with a quote proposal based on the number of plasmids required, their complexity, the number of synthesis needed, and the amplification scale asked.

Submit your request using our contact us form. A first ballpark estimation will be rapidly sent to you. Based on the requested plasmid quantity and quality grade, a detailed quotation will be edited including the adapted strategy, the successive work packages and deliverables, the timing as well as the associated budget.

The turnaround time for the manufacturing of one R&D grade batch ranges between 4 and 6 weeks depending on the existence or not of a cell bank. The timing required for the manufacturing of a set of 4 plasmids will take between 9 and 11 weeks. Ten additional weeks are necessary for the release of one GMP batch.

The maximum quantity of plasmid that can be delivered is 20 g regardless of the quality grade considered (R&D, HQ or GMP) We also offer a pDNA amplification service for quantities up to 50 mg at a discovery grade

A project of plasmid production can be performed using either a cell bank generated by Xpress Biologics or a cell bank provided by the customer. For biosafety purpose, the bank provided by the customer must be supplied with a CoA attesting the absence of bacteriophages.

HQ grade plasmid can be used as a drug substance for preclinical trials or as starting material for preclinical and clinical phases I/II trials. The produced and purified plasmid is characterized using a complete set of quality controls covering attributes related to physical state, identity, content, purity/impurity and safety. 

The GMP grade is required when the plasmid is used as a drug substance for all clinical trials and as starting material for clinical phases II/III trials. (Voir figure dans section “overview”). The produced and purified plasmid is characterized using a complete set of quality controls covering attributes related to physical state, identity, content, purity/impurity and safety. (Voir tableau ci-dessous: List of quality controls for the different quality grades of plasmids). The majority of the tests are compendial and therefore . Qualification of some specific tests such as the plasmid topology by HPLC has to be considered and is proposed by Xpress Biologics.

Inverted Terminal Repeats or ITR sequences that are present in AAV vectors are needed to guide genome replication and packaging during vector production. The ITR sequences usually form hairpin structures, which modifies the charge density of the plasmid leading to a different chromatographic behavior potentially decreasing the efficiency of the purification process. This is due to the fact that the presence of the ITR sequences renders the charge density of the supercoiled (SC) form similar to that of the open-circular (OC) form. The chromatographic conditions have been therefore modified in order to successfully separate both isoforms and allow the recovery of a proportion of SC form conform to the specification (˃95%).

The presence of repetitive sequences (ex: LTR sequences in LV vectors) could promote recombination events during the amplification phase. Xpress Biologics has developed a fermentation process avoiding such recombination events while allowing a plasmid yield at harvest compatible with industrial and economical constraints. Moreover, our analytical methods allow the complete sequencing of plasmids containing repetitive sequences and therefore can verify the absence of potential recombination.

Over the last 3 years, Xpress Biologics has managed around thirty manufacturing projects of plasmids dedicated to LV production, which corresponds to more than fifty batches, including R&D, HQ and GMP grade lots.

Over the last 3 years, Xpress Biologics has managed around twenty manufacturing projects of plasmids dedicated to AAV production, which corresponds to thirty batches, including R&D and HQ grade lots.

Xpress Biologics has adapted its manufacturing process (mainly fermentation) in order to take into account the presence of the long poly(A) tail in the plasmid sequence, tail up to 120 nucleotides being successfully produced and purified. When linearization is requested by the customer, a feasibility study is proposed in order to optimize the digestion conditions, with the aim to limit the amount of restrictive enzyme and therefore the production budget.

Due to the high degree of purity achieved following the purification protocol developed by Xpress Biologics (absence of nucleases), the stability of the plasmids appears to be excellent not only when the plasmid is frozen at -20 or -80°C (stability of more than 3 years), but also when stored at 4°C or RT. Moreover, based on the extensive experience acquired in recent years, Xpress Biologics can advise the customer on the nature of the formulation buffer to be used to ensure long-term stability. If required to support for instance the submission of a dossier for the introduction of a new drug, an extended stability study can be initiated following the ICH guidelines.

Yes, we offers a service of plasmid engineering based on an innovative assembly technology (e-Zyvec^®). Our online software and dedicated plasmid experts can help you to define the plasmid features that fit to your application and that are compatible with large scale manufacturing requirements.