DNA vaccines offer several advantages over traditional vaccine production methods: 1) They are faster to produce, taking 4-6 weeks from gene sequence to final vaccine compared to 6 months for traditional methods. 2) They are less expensive and use simpler production processes than traditional or recombinant protein vaccines. 3) Early safety studies have not found DNA integration into chromosomes as some initially feared. However, DNA vaccine development has faced challenges and it has taken nearly 20 years to optimize the technology for human use. Significant progress has been made in manufacturing scale-up and ensuring product stability. If clinical trials continue to show DNA vaccines are safe and effective for humans, it could revolutionize vaccine production.

1. V a c c i n e s section one
DNA Vaccine Technology
Biotechnology’s Brave New World
by Magda Marquet
P
icture rows and rows of for a production run down to weeks.
chicken eggs incubating not to Last year, the industry managed to
hatch chickens, but to produce produce H1N1 flu vaccines in about
vaccines. With the exception of 30–40 weeks, but that wasn’t good
a few products on the market now, enough. DNA vaccines take four to six
most vaccines are still made using this weeks to go from gene sequence to vial.
50-year-old technology. Using chicken The production process is safer, as are
eggs to produce vaccines takes about the products themselves. We’ve closely research started. A 1993 paper
half a year to complete and requires examined the potential issue of DNA reported on a collaboration between
on average one to two eggs to make a integration into the chromosomes of Merck and Vical showing that the
single vaccine dose. It is inefficient, recipients. In many safety studies, we’ve technology worked quite efficiently in
labor intensive, time consuming, and never found that to happen. The an influenza model (2). In 1994,
subject to contamination. The latter product doesn’t integrate, and it gets David Weiner at University of
may be the most important problem. In removed from a person’s body over time. Pennsylvania filed the first IND for a
addition to the vaccine you want, DNA vaccine technology is also DNA vaccine. And then my group at
contaminants (including other viruses) less expensive than that for traditional Vical developed the first processes to
can get into the product stream. vaccines. It uses bacterial fermentors make pharmaceutical-grade DNA (3).
But there may be a way to that can be used for other processes, When I entered the field, all the
modernize our vaccine technology. and DNA vaccines can be made using methods used for DNA production
Rather than producing a virus or a very simple techniques — even more were based on the use of ethidium-
protein to inject into an organism, we straightforward than the recombinant bromide double-cesium purification,
can produce a sequence of DNA that DNA technologies used to express resulting in DNA that could not be
has been genetically engineered to protein subunits. injected into humans. So we needed a
express proteins from a specific However, all the market successes process that could be scaled up for
pathogen when introduced into an we’ve had so far have been in animals: clinical (and eventually commercial)
organism. The idea is that once the horses, salmon, pigs, and dogs (Table vaccine manufacturing. Although now
organism’s cells have made the proteins 1). The dog melanoma vaccine, in it’s pretty obvious to purify kilogram
from that DNA sequence, the immune particular, has made a big difference in quantities of DNA, fifteen years ago it
system will recognize them as foreign curing a problem that was previously required a change of mind set for
and launch a response against them. very difficult to treat. This validates biochemists who were used to purifying
I’ve been working in DNA vaccines the DNA vaccine technology, and it protein and getting rid of DNA.
for two decades. At first, as strong also shows that we’re almost ready for The early 1990s generated a lot of
believers in a powerful technology, we human vaccines. It demonstrates that excitement about DNA vaccines,
thought we would have approval in this technology is very cost efficient followed by some clinical
humans in less than five years — but because costs in veterinary applications disappointments — except in animals,
that didn’t happen. Yet this is still a are necessarily lower than in human where the technology has worked very
field that I believe will revolutionize the medicine. well. Since then, we’ve seen
world of vaccines. We still have many refinement and optimization of DNA
challenges to overcome, but it’s clear hisTorical oVerView vaccine expression, potency, and
that we have made significant progress. In 1990, by pure serendipity Wolf and immunogenicity. Novel formulations
Felgner discovered in a controlled and adjuvants are providing much
Technological success experiment that injecting DNA in better protection. There’s been
Why use DNA vaccines? First, they are mice produced an immune response optimization at the genetic level: e.g.,
fast: decreasing the traditional months (1). That got the field of DNA vaccine codon optimization to increase T-cell
2 BioProcess International 9(8)s S eptember 2011 Supplement

2. Table 1: Four veterinary vaccines have been licensed, which validates the technology.
response. And heterologous prime-
Target Company/Sponsor Year Approved Species
boost vaccination is making
West Nile virus Centers for Disease Control and 2005 Horse
significant inroads (4). Harriet Fort Dodge Laboratories
Robinson’s group at Emory University Infectious heamatopoietic Novartis 2005 Salmon
in Atlanta, GA, has achieved good necrosis virus
results in primates using DNA for Growth-hormone–releasing VGX Animal Health 2007 Swine
priming and recombinant modified hormone
vaccinia Ankara (MVA) virus. Melanoma Merial/MSK 2007 Dog
ManufacTuring Progress Figure 1: Fermentation optimization (plasmid yield improvement)
Meanwhile, diligent work in
1,200
manufacturing technologies has
addressed yield and stability issues. 1,067.00
These methods are scaling up
1,000
successfully to at least 1,000 L or more.
And a 1,000-L process can yield 866.40
Plasmid DNA (mg/L)
millions of vaccine doses, which allows 800
for commercial production in a relatively
small plant. Disposable bioreactors do 652.00
not work for DNA vaccine production 600
because of oxygen-transfer limitations. Original Batch Process
But single-use technologies do play a
381.23
significant role in downstream 400
374.92
processing to reduce cleaning validation
work, improve manufacturing 186.15
efficiencies, and lower overall costs. 200
119.72
Major progress in formulation and 75.79
54.00 62.56
85.50
61.90
82.33
lyophilization technologies is making 13.54
0
DNA vaccines more shelf stable. The Fed-Batch Development Experiments
cold chain is a major issue for all
vaccines, especially those sent to
2 Ulmer, et al. Heterologous Protection
developing countries. Not everyone a BraVe new world Against Influenza By Injection of DNA
has the necessary refrigeration or Are DNA vaccines ready for prime Encoding a Viral Protein. Science 259, 1993.
freezing equipment to store sensitive time (5)? It’s taken nearly 20 years, 3 Horn, Marquet, et al. Cancer Gene
liquid formulations. and development is ongoing. Things Therapy Using Plasmid DNA: Purification of
Case Study: Figure 1 shows the didn’t work the way we initially DNA for Human Clinical Trials. Hum. Gene
kinds of product yields we’ve had over imagined they would, but that’s Ther. 6, 1995.
the years at Althea. It begins with similar to the story of monoclonal 4 Robinson HL. Prime Boost Vaccines
Power Up in People. Nat. Med. 9, 2003: 642–643.
something we thought was pretty antibodies (MAbs). After 20 years, by
5 Kutzler MA, Weiner DB. DNA
good at the time! Now we regularly the time many investors didn’t believe Vaccines: Ready for Prime Time? Nat. Rev.
Genetics 9, 2008: 776–788. c
see g/L product titers. Human-vaccine in MAbs anymore, the technology
lots we’ve produced range mainly from finally worked — and that changed
preclinical to phase 2 scale. We’ve the pharmaceutical industry. I see this
Magda Marquet, PhD, is cofounder and
worked with clients from Europe, the coming for DNA vaccines as well. cochair of the board of directors for Althea
United States, and the Asia–Pacific Major progress has been made not technologies, inc., 11040 Roselle street, san
region, to produce preclinical, phase 1, only in basic science and product Diego, cA 92121; 1-858-882-0123, fax 1-858-
and phase 2 products. development, but also in delivery and 882-0133; mmarquet@altheatech.com.
Our process development team has manufacturing. Collaboration among
progressively been able to increase industry, academic laboratories, and this article is adapted from a presentation
manufacturing yields through a range governments has facilitated much of given at Interphex in new York, nY, March
of fermentors from the 30-L scale to that progress. Clinical trials are 2011 in a lunchtime session moderated by
the 100-L scale and now at the 1,000- ongoing to assess expression, potency, BPi’s editor in chief, s. Anne Montgomery.
L level. We’re still working in the and immunogenicity. Once we get one
clinic to make sure that we have the success, then many others will follow,
To order reprints of this article, contact
right human-vaccine product before and it will be a revolution. Rhonda Brown (rhondab@fosterprinting.com)
we can go to market. But in terms of 1-800-382-0808. Download a low-resolution
manufacturing, we’re ready. That’s not references PDF online at www.bioprocessintl.com.
the limiting step. 1 Wolf, Felgner, et al. Direct Gene Transfer
into Mouse Muscle In Vivo. Science 247, 1990.
Supplement S eptember 2011 9(8)s BioProcess International 3