DNA Introduced Directly Into Cell Nucleus Using Protein Nanodisks

These protein nanodisks are similar to the minivector DNAs developed by Baylor College of Medicine in that they have improved transfection efficiency and increased serum survival.

http://www.sciencedaily.com/releases/2011/01/110111132529.htm

IL-13Rα2 can serve as a potent tumor antigen that can recruit immune responses against IL-13Rα2 expressing solid tumors

This study evaluated prophylactic and therapeutic effects of the IL-13Rα2 cDNA vaccination in syngeneic animal models of melanoma, sarcoma and breast cancer cells expressing IL-13Rα2 to prime the immune system. CTL activity and IFN-γ release was measured in mice vaccinated with prophylactic IL-13Rα2 DNA and boosted with ECDα2, resulting in higher measurements than DNA alone. Therapeutic IL-13Rα2 DNA and boost vaccination inhibited established MCA304, 4T1, and D5α2 tumor growth. Overall, immunization with IL-13Rα2 DNA vaccine followed by ECDα2 boost mixed with CpG and IFA adjuvants inhibits tumor growth and significantly prolongs the survival of mice compared to DNA vaccine alone.

Hideyuki Nakashima, Toshio Fujisawa, Syed R Husain, Raj K Puri.
“Interleukin-13 receptor α2 DNA prime boost vaccine induces tumor immunity in murine tumor models.”
Journal of Translational Medicine 2010

DNA prime/mixed VLP vaccination shows promise against HIV-1

This study confirms that enhanced cellular immune responses result in mice via chimaeric Gag VLPs incorporating GagRT and GagTN as vaccine boost candidates to a pVRCgrttnC DNA priming vaccine for HIV-1. The DNA prime/mixed VLP boost vaccination increased the cumulative Gag- and RT-specific IFN-γ responses relative to two DNA vaccinations 4-fold, and Gag- and RT-specific IL-2 responses 2.8 fold. Chimaeric VLP boosts such as these are a promising option for future HIV-1 vaccine studies.

Sirika Pillay, Enid G Shephard, Ann E Meyers, Anna-Lise Williamson, Edward P Rybicki.
“HIV-1 sub-type C chimaeric VLPs boost celluar immune responses in mice.”
Journal of Immune Based Therapies and Vaccines 2010.

Plasmid DNA 101

A plasmid is extra chromosomal DNA that is not incorporated in the main helix of genomic DNA. Plasmids occur in nature and can be found everywhere – in plants, insects, and bacteria. Scientists have discovered various applications for plasmids including gene therapy and DNA vaccines. These applications deliver the plasmid to a specific target in an organism to obtain a desired response. Plasmids can be delivered as a naked molecule or encapsulated in a nanoparticle. Other delivery methods are being developed to improve plasmid delivery efficiency and increase serum survival lengths (see Minivector DNAs).

Most plasmids are designed to contain an origin of replication, a gene of interest and a gene for antibiotic resistance. In a process called cloning, a gene is inserted into an empty plasmid backbone (vector) using genetic engineering. Plasmids are specifically designed to replicate in bacteria without gene expression; yet, in their intended target, they express the gene without replication.                  

Once a plasmid is assembled with the proper components, it is transformed into a competent cell. In this process, electrical current or chemistry opens the bacterial pores to accept the plasmid, and the competent cells end up with several copies of the plasmid. To isolate only the plasmid-containing bacteria, the cells are cultured with the antibiotic corresponding to their antibiotic resistant gene.

 Since the plasmid includes a resistance marker to the antibiotic, a cell that accepts the plasmid will be resistant to the antibiotic. Cells that reject the plasmid will not be resistant and will be instantly killed. The resistant cells are frozen as the master cell bank (MCB), and the MCB is used to make a working cell bank (WCB). Upon completion of cell banking, the WCB is then fermented to replicate enough plasmid for use in animal studies and clinical trials.

-Henry H.

Improved gene silencing capabilities with minivector DNAs

Baylor College of Medicine, in conjunction with The Methodist Hospital Research Institute, has developed a way to use minivector DNAs in gene therapy. These reduced sized DNA vectors (as small as 300bp) have high gene silencing capabilities and high serum survival lengths with low replenishment requirements. In addition, the study shows that DNA minivectors have improved transfection efficiencies in aortic smooth muscle and NIH 3T3 cells. Minivector DNAs are a promising new gene therapy tool, particularly for blood-borne diseases such as lymphoma.

Zhao, N., J M Fogg, L Zechiedrich, and Y Zu.
“Transfection of shRNA-encoding Minivector DNA of a few hundred base pairs to regulate gene expression in lymphoma cells.”  Gene Therapy  21 October 2010.



Investing for the Future: Quality cGMP Cell Banks

Cell banking is essential for a biological product manufactured by the fermentation process, such as plasmid DNA. Particular emphasis is given to cGMP cell banks by following the regulatory standards (21CFR610). But how do you generate high quality cGMP cell banks, and what are considered important attributes? Guidelines do not tell us.

First, let’s look at the two-tiered bacterial cell banking system:

• Seed stock –> Master cell bank (MCB) –> Working cell bank (WCB)

Seed stock is established from a single colony (or clone), MCB is generated from the seed stock to ensure the genetic integrity within cells, and WCB is produced from MCB and used for production. A multitude of characterization assays are then performed, such as identity, purity, phage, cell viability, plasmid retention, copy number, sequencing, restriction analysis, etc.

Although the process seems straightforward, is it sufficient to simply pick a single colony and grow it up for use as an MCB and WCB? No. (Believe it or not, that is what most research and manufacturing organizations do).

It’s important to perform extensive screening on the seed stock prior to generating cGMP cell banks. Additional screening on the seed stock will directly impact the quality of the final cGMP product. Below is the VGXI cell banking system:

• Seed stock –> Screening –> Master Cell Bank (MCB) –> Working Cell Bank (WCB)

Why extra screening? Two simple reasons: Quality and Quantity.

Quality

A plasmid may assume dynamic structural conformations: supercoiled (SC), open circular (OC), relaxed, linear, dimer, multimer, deletion, etc. The quality of a plasmid is generally acknowledged as the SC percentage, or more specifically, the SC monomer percentage. Therefore, rational and efficient design of the vector sequence is vital. Dimer and multimer forms for a plasmid of large size or with repetitive DNA sequences have been frequently observed. During the screening process, colonies of pure dimers or deletions are able to be eliminated. Even if all selected colonies assume correct size, colonies can be screened for high SC monomer percentage.

Quantity

Plasmid yield, or copy number of MCB/WCB, will affect the production process. Yet, based on experience, plasmid copy number of individual colonies is not solely controlled by the replication of origin, and not all host/vector systems have homogeneous copy numbers for all single colonies. So regarding the quantity of your cell banks, you may need to answer two questions:

(1) Did I pick the colony of the best yield (shaker flasks)?

(2) Will I still get the highest yield in fermentation (bioreactors)?

Over 4-fold yield differences among single colonies have been observed when tested in shaker flasks. Furthermore, yields in shaker flasks frequently do not correlate with high cell density fermentation in the bioreactors. For one plasmid tested, 2-3 fold differences in specific and volumetric yields were shown for two seed stocks which had similar yields in shaker flasks.

Overall, generating high quality cGMP cell banks requires a lot of work but is an investment for the future; it will demand less optimization of the fermentation process and improve product purity for downstream processing.

-Ying C.

Helpful GMP Tips for the QA Manager

As a QA Manager for a plasmid manufacturer, I am always looking for innovative ways to maintain a focus on GMP and help employees understand its importance. GMP training accomplishes this, but it only occurs once or twice a year in most organizations. If you do not have a dedicated training department, it most likely only occurs once a year.

I devised a simple way to provide GMP information to employees on a more frequent basis. It is called the GMP Newsletter. It is published once a month by QC and may include information on GMP regulations or general GMP related topics. The newsletter requires an employee signature to document that the employee has read and understood the newsletter, and it is filed in their training binder.

The newsletters we have issued have covered a vast amount of subjects. One of the most interesting was a series of articles on the history of GMP regulations. It included a timetable for the implementation of the statutes and highlighted significant events that led to certain ones (i.e. Upton Sinclair’s book The Jungle, the Tylenol scare, etc.).

Other content included audit preparation, good documentation practices, investigating human error, and even one consisted of a crossword puzzle with terms frequently used in a Quality System. Topics can be almost anything.

The idea is to keep a continuous focus on Quality and GMP by providing information on a more periodic basis, and a monthly newsletter has proven to be an effective way of doing that!

– Dorothy P.

AIRMIX – A Softer, Gentler Lysis

E. coli cell paste.

Arguably, the most challenging step in plasmid manufacturing is cell lysis. Thousands of times larger than a typical small molecule pharmaceutical API, a plasmid is a large circular molecule that most of our clients prefer in the supercoiled form.

Several things can go wrong if the lysis step is too harsh. First, the plasmid molecule will shear or unwind to form an open circle. Second, the host cell’s genomic DNA will shear making it difficult to separate from the plasmid.

Knowing this, VGXI spent the first 2-3 years of operations developing its patented lysis process. The process had to be gentle enough to not shear the plasmid or genomic DNA but stringent enough to break open the cells and separate most of the cellular debris and genomic DNA from the plasmid. Using conventional methods (manual lysis by hand), a 50 kg batch of cell paste would have taken 3-4 months to lyse. Our goal was to produce an automated process that could lyse 50 kg of cells in less than a day.

The process we developed employs tanks connected in series that perform different mixing and filtration steps. There are a couple of key mixers in the machine, such as our patented “bubble column” mixer. This is a long tube full of bubbles that the cell lysate runs through. The bubbles gently separate the lysed cellular components without shearing the plasmid or genomic DNA. As the cell solution runs through the bubble column, special chemicals in the lysis solution cause the genomic DNA to tangle with the cellular debris. The cell debris and genomic DNA then float to the top of the lysis solution and form a thick, oatmeal-like cake. The solution containing the plasmid is pulled off the bottom of the tank for further processing.

Where it used to take 2-3 days to lyse a large scale batch, it now takes only a few hours. Furthermore, VGXI’s patented bubble mixer removes over 99% of cellular and genomic DNA contaminates simplifying downstream purification.

-Henry H.

Minimizing Cleaning Validation

Preparing for large scale plasmid purification.

One of the major challenges of plasmid manufacturing is cleaning validation. To avoid excess costs and time, disposable technology can decrease validation needs for both the cleaning and sterilization processes of fixed equipment systems.

Disposable materials for use in pharmaceutical manufacturing have come a long way from traditional reusable technology. From pipettes and syringes to disposable bioreactors and pre-packed purification columns, disposable single-use materials have many advantages for pharmaceutical manufacturing, including:

• Low startup cost
• Pre-sterilized components and assemblies
• Plug-and-play operations
• Increased flexibility in design and operation of systems
• Reduced validation time and cost
• Reduced changeover time

These advantages have stimulated the rapid adoption of disposable technology in clinical and small scale production applications. Single-use disposables, today, include flexible tubing systems with either direct aseptic connections or tubing fusing systems, process mixing containers, large scale bioprocess reactors, and sensors for the measurement of process parameters.

The advances in single-use disposable materials are making it easier for the Contract Manufacturer to meet the diverse and exacting requirements of researchers and gene based therapy companies in need of additional processing capabilities.

-Jerry B.

From Dreadful to Enjoyable: Transform Your GMP Training

Given the right setting, GMP training can be fun and interesting.

Most companies struggle with how to make GMP training more fun and exciting. As the Quality Assurance director of a company that provides contract plasmid manufacturing, it seems almost impossible for me to find time to be creative.

This year for the “dreaded” GMP training we played a game – yes, a game – called Wheel of Compliance.  This training was a “spin-off” of Wheel of Fortune.  We had a wheel and everything!  A very colorful one I might add.

The employees were split up into 3 teams, and, one by one, a player from each team spun the tri-colored wheel.  The color the ticker landed on determined the difficulty of the question.  To encourage learning and participation, points were only awarded for CORRECT answers and prizes were given to the winning team.

Trust me, games bring out competitive spirit and, therefore, are very effective learning tools.  Everyone listens to ALL the questions to test their own knowledge.  It is an interactive, fun way to learn!

As an annual tradition, we invent a new game for GMP Training Day.  In 2008, we played GMP Baseball.  It was Team Plasmids vs. the DNA Vaccines. Participants actually ran the bases for correct answers.  Questions corresponded to the types of hits and were based on difficulty level (i.e. the question related to a single is easier than the question for a homerun).  In 2009, we played Jeopardy.  Both regular and double Jeopardy rounds were included as well as a Final Jeopardy question!

So, get out there and create innovative training sessions!

The usual groans and yawns that accompany the formidable training day will soon be transformed into laughs and competitive spirit.

If you have any great ideas, please share them in the comment section below!

– Dorothy P.