Biopunk: DIY Scientists Hack the Software of Life

I just got my copy of the book “Biopunk:  DIY Scientists Hack the Software of Life“ and it’s a page turner: pithy and fascinating.  It also features a couple chapters on my LavaAmp co-conspirators Guido and Joseph. We’re getting some decent free press on this one, that’s for sure. There was a feature last week in The Guardian interviewing Joseph and Guido won us a $40,000 USD grant through Start Up Chile.

From a write up in BoingBoing in April comes this quote: “We reject the popular perception that science is only done in million-dollar university, government, or corporate labs; we assert that the right of freedom of inquiry, to do research and pursue understanding under one’s own direction, is as fundamental a right as that of free speech or freedom of religion,” Patterson writes in A Biopunk Manifesto, a biohacker call-to-arms she wrote last year.

“We have no quarrel with Big Science; we merely recall that Small Science has always been just as critical to the development of the body of human knowledge, and we refuse to see it extinguished.”

That’s a “Two Fer” Meredith.  As in fer shure, fer shure……..

BioBeers Friday, Badgers, News and other Random Stuff

As if I haven’t pestered you all anough already BioBeers is this Friday at ImQuest Biosciences Friday February 18th starting at 4:30 PM. I am getting Ribs from RibCity, so please do RSVP so I can get the right amount. RSVP’s trickling in thus far and I am giving you multiple choices. You can RSVP by emailing me, leaving a comment or go on the MeetUp or LinkedIn sites (both require FREE registration). Badger, badger as in “Her hungry fingers tore at my shirt buttons like wild badgers” (a hilarious episode of Prairie Home Companion I listened to on the way back from Fredericksburg VA last week http://is.gd/UWzJWF can’t help it that I am a radio junkie). 

Speaking of Badgers, Akonni was in the news this week for signing a licensing agreement with USAMRIID which covers covers nucleic acid sequences, primers, and probes that will serve as the basis for multiplexed molecular tests for Bacillus anthracis, vaccinia/orthopox virus, Yersinia pestis, and Venezuelan equine encephalitis virus. How neat is that?

Speaking of neat stuff, you all may know that I have been working on an inexpensive, hand held thermocycler (LavaAmp).  Well, so compatriots of ours, Tito Janokoswski and crew with pearl biotech, have released a DIY $500 thermocycler.  It’s based on the standard tube format PCR in aluminum block, so I don’t consider this a threat at all to our low resource, low power, portable device, but cool news nonetheless out of the DIYBio guys.  They also have the Open Gel Box, which I contributed to.  Keep it up!

So back to FredCoBio and stuff happening here BioElectronics is poised for huge growth.  I know I haven’t blogged about them in a while, but their patch works and it’s not expensive and it’s really the only thing out there.  I think it’s awesome!!

The Near Eastern Origin of Cat Domestication in Fredcobio

I am always amazed at the breadth and depth of scientific research going down in our own back yard. I bet you didn’t know that this 2007 paper in Science was partially done in Frederick: The Near Eastern Origin of Cat Domestication

A few weeks back I went to Olive’s, on a whim, and ended up chatting with this cat Carlos who works at the Laboratory of Genomic Diversity at NCI over a martini or two.  Fascinating stuff.   The research shows that wildcats (Felis silvestris) were initially domesticated in the Near East, probably coincident with agricultural village development in the Fertile Crescent.  Interestingly, cats differ from other domesticated animals in that they seem to provide no real benefit to humans aside from catching rodents in the early grain stores & companionship.

The Science text is a very detailed technical account and is followed up with a more elaborate, “scientifically toned down” article in the June 2009 edition of  Scientific American.

Some conclusions of this article

Unlike other domesticated creatures, the house cat contributes little to human survival.  Researchers have therefore wondered how and why cats came to live among people.

Experts traditionally thought that the Egyptians were the first to domesticate the cat, some 3,600 years ago. But recent genetic and archaeological discoveries indicate that cat domestication began in the Fertile Crescent, perhaps around 10,000 years ago, when agriculture was getting under way.

The findings suggest that cats started making themselves at home around people to take advantage of the mice and food scraps found in their settlements.

In an article published in PNAS in 2009, the authors delve into the differences between Natural vs. Artificial Selection.  Interesting stuff:

We perceive today, as did Darwin, that natural selection is the environmentally driven mechanistic process by which more advantageous traits are, on the whole, passed on to succeeding generations more often than less advantageous traits because of differential reproduction of the individuals possessing them. Sexual selection is a natural process of intraspecific competition for mating rights. Artificial selection, generally the motive force behind domestication, is often equated with selective breeding. This often amounts to prezygotic selection (where mates are chosen by humans) versus postzygotic selection (where the most fit progeny reproduce differentially) as in natural selection. Although natural selection plays a considerable role in the evolution of many traits (e.g., disease resistance) during the animal domestication process, sexual selection is effectively trumped by the human-imposed arrangements of matings and often by the human desire for particular secondary sexual characters. Artificial selection is a conscious, if unintentional, process, and therefore is generally considered to be effected only by humans.

This is exactly the kind of research we’re doing at Ft Detrick that I think would be brilliant to get out to people by a yet to be established mechanism. I wonder if The various agency “partners” at Ft Detrick even track this stuff?

Eppendorf to market Akonni TruTip™

Just saw this run across the wire.  Sounds like a great deal for Akonni and FredcoBio:

Eppendorf North America and Akonni Biosystems Announce Joint Marketing Agreement

Eppendorf NA to market Akonni TruTip™ solutions for rapid, high-throughput nucleic acid extraction on epMotion^® automated pipetting systems

 HAUPPAUGE, N.Y. & FREDERICK, Md.–(BUSINESS WIRE)–Eppendorf North America and Akonni Biosystems today announced they have entered into a joint marketing agreement to promote Akonni TruTip nucleic acid extraction kits configured for use with Eppendorf epMotion automated pipetting systems. Under the agreement, Eppendorf will promote the extraction kits to clinical, clinical research and forensic laboratories in North America – providing users with access to the industry’s most rapid and reliable means to automatically extract PCR-ready DNA and/or RNA from larger volume samples.

“Eppendorf’s deep experience in developing and manufacturing automated pipetting systems makes them an excellent partner for our high-throughput TruTip nucleic acid extraction solutions”

“Eppendorf’s deep experience in developing and manufacturing automated pipetting systems makes them an excellent partner for our high-throughput TruTip nucleic acid extraction solutions,” said Kevin Banks, Ph.D., Vice President of Sales and Marketing at Akonni. “We believe that the combination of the products from both companies has the potential to improve laboratory efficiency by reducing, by 10-fold or more, the time it takes to obtain PCR-ready nucleic acid, for up to 8 simultaneous samples in as few as 4 minutes.”

Andrea Dickstein, Eppendorf North America Director of Marketing, adds, “It is well known that rapid and reliable nucleic acid extraction is a critical early step in detecting diseases and genetic disorders using genomic-based methods, like real time PCR. By combining the rapid extraction methodology used in TruTip with epMotion’s automated pipetting systems, we’ll increase the productivity of our customers by reducing to mere minutes the time it takes to extract DNA or RNA.”

Akonni TruTip uses a patented, nucleic acid binding matrix inserted into the 1000µl Eppendorf pipette tip for the epMotion system. Each TruTip Kit delivered under this agreement includes a rack of 96 x 1000µl TruTip extraction tips with bulk packaged lysis, wash and elution buffers.

Akonni will initially introduce two high-throughput TruTip extraction kits for use on the epMotion systems: one for isolating Influenza RNA from nasopharyngeal aspirate samples, and a second for extracting genomic DNA from saliva. Akonni plans to develop and supply additional kits for the Eppendorf epMotion family of systems in the near future.

Opportunities Abound

As you may already be aware, one of the primary reasons the National Cancer Institute is building the new Riverside Research Park is to provide space for “synergistic partners” from academia and Industry to work together to cure cancer.  I was just alerted to several new opportunities by my friends at FITCI

A new collaboration opportunity, “Gene Expression Signature Predictive of Response to Chemotherapy” has been added to the NCI Technology Transfer Center web site. Please go to: http://ttc.nci.nih.gov/opportunities/opportunity.php?opp_id=1881

A new collaboration opportunity, “Antibody and Immunotoxin Treatments for Mesothelin-Expressing Cancers” has been added to the NCI Technology Transfer Center web site. Please go to: http://ttc.nci.nih.gov/opportunities/opportunity.php?opp_id=1883

A new collaboration opportunity, “Knockdown and Enhanced Expression of P53 Isoforms to Treat Age-Related Disorders and Cancer” has been added to the NCI Technology Transfer Center web site. Please go to: http://ttc.nci.nih.gov/opportunities/opportunity.php?opp_id=1885

A new collaboration opportunity, “Engineered Biological Pacemakers” has been added to the NCI Technology Transfer Center web site. Please go to: http://ttc.nci.nih.gov/opportunities/opportunity.php?opp_id=1884

A new collaboration opportunity, “Novel Kinase Inhibitors Targeting the PH Domain of AKT for Preventing and Treating Cancer” has been added to the NCI Technology Transfer Center web site. Please go to: http://ttc.nci.nih.gov/opportunities/opportunity.php?opp_id=1882

LavaAmp Launched Coast to Coast

One reason I have been lame in the blogging department is that I have been working on the LavaAmp™ project.  After SciFoo camp last year, I was asked by Joseph Jackson and Guido Nuñez-Mujica if I  could help them licences this device from Texas A&M.

Guido is from Venezuela and is most interested in infectious disease testng in 3rd world, remote applications.  This is a talk Guido gave at Google shortly before SciFoo camp:

After nearly a year of negotiating with the TAMU tech transfer office (some day I’ll blog about University Tech Transfer offices stifling Innovation and commercialization) in the past 6 weeks we recruited Rob Carlson and Rik Wehbring from Biodesic and a the engineering prototype is built.

A more technical description from Robs blog post:

“The LavaAmp is based on the convective PCR thermocycler demonstrated by Agrawal et al, which has been licensed from Texas A&M University to Gahaga.  Under contract from Gahaga, Biodesic reduced the material costs and power consumption of the device.  We started by switching from the aluminum block heaters in the original device (expensive) to thin film heaters printed on plastic.  A photo of the engineering prototype is below (inset shows a cell phone for scale).  PCR reagents, as in the original demonstration, are contained in a PFTE loop slid over the heater core.  Only one loop is shown for demonstration purposes, though clearly the capacity is much larger.”

So we’re off and running.  Joseph is out at BilPil in San Diego this weekend with the device and hoping to get a little mention in at iGEM Jamboree in Boston, although it’s hard to be in two places at once.

A bit more from Synthesis:  ”The existing prototype has three independently controllable heating zones that can reach 100C.  The device can be powered either by a USB connection or an AC adapter (or batteries, if desired).  The USB connection is primarily used for power, but is also used to program the temperature setpoints for each zone.  The design is intended to accommodate additional measurement capability such as real-time fluorescence monitoring.

We searched hard for the right materials to form the heaters and thin film conductive inks are a definite win.  They heat very quickly and have almost zero thermal mass.  The prototype, for example, uses approximately 2W whereas the battery-operated device in the original publication used around 6W.

What we have produced is an engineering prototype to demonstrate materials and controls — the form factor will certainly be different in production.  It may look something like a soda can, though I think we could probably fit the whole thing inside a 100ml centrifuge tube.”

And Attila over at PIMM also beat me to the blog punch.

I need to put together the press release and finish plans for BioBeers on Friday,  put more marketing material in our Business Plan and write up a new proposal for amniotic tissue skin grafts this morning.  Gaining momentum…..

My Delight, but a Germophobes Nightmare

I am always (perhaps too much) scanning the blogosphere for anything related to FredCoBio news.  This story I predict will make the Network news tonight or maybe tomorrow.

The entire article is published in Science (and will require a paid subscription to view your tax dollars at work), but ther have been a number of AP stories circulating on the blogesphere today. You can read the whole story at ScienceNOW:

Your Body Is a Wonderland … of Bacteria

By Stephanie Pappas
ScienceNOW Daily News
28 May 2009

Where can you find your skin’s most diverse community of bacteria? Not in a sweaty armpit or linty belly button. According to a new survey of the bacterial ecosystem that covers us, the diversity hot spot of the body’s exterior is the forearm. And the surprises don’t end there.

Microbes that live in and on our bodies outnumber our own cells 10 to one, but researchers have only recently begun to catalog the residents on our skin. Traditionally, scientists identified human skin bacteria by swabbing volunteers and culturing the samples, but those results skewed toward microbes that grow well in the lab. Thanks to ever-evolving gene-sequencing technology, scientists can now use microbial RNA to identify organisms. With these techniques, researchers have found an unexpectedly wide variety of bacteria on human skin (Science, 23 May 2008, p. 1001). But no one had ever systematically compared bacterial colonies from different areas on the human body.

To do so, scientists from the National Human Genome Research Institute in Bethesda, Maryland, recruited 10 volunteers and asked them to wash with mild soap for 1 week. Then, after 24 hours without bathing, the volunteers arrived at the lab, where researchers swabbed and scraped their skin in 20 places–everywhere from the nostril to the navel to that bane of low-rise jeans aficionados, the gluteal crease. The team analyzed ribosomal RNA from the samples and classified the microbes based on their genomes.

The researchers found about 1000 species total, which were fairly consistent from person to person; it turns out we all have similar tenants in our noses and on our backs. The number suggests that our skin is as variegated as our guts, which house anywhere from 500 to 1000 bacterial species. The team also found vast differences across the skin, according to the study published in tomorrow’s issue of Science. Contrary to what acne-prone teenagers might expect, oily areas such as the forehead and scalp are actually less diverse than dry areas such as the forearm (though one is enough for grief: Propionibacterium acnes thrives in oily spots). The most barren region was behind the ear, with a median diversity of 15 species. In comparison, the forearm teemed with a median 44 species. A follow-up with five of the volunteers months later found that bacterial makeup changed little over time.

The link to FredCoBio is simple. Alice Y. and I worked together in at Molecular Diagnostic Division of Life Tech, which is now known as Digene/Qiagen.  She used to give us so many clothes passed down from her daughter, Rachel, to our oldest (now 20, yikes!).  And Bob B. is an original BRL guy who lives in Frederick.  I remember his wife worked the cash register at Martins on 7th street when we used to live in the city.  Anyway, they are both at NHGRI now, along with a number of other former Life Tech people.

Pretty cool research and yet another deep tie into the FredCoBio community.

Did I forget to mention that LifeTech is expanding in Frederick?

I read yet another story in an out of town publication about the expansion at LifeTech in Frederick.

I case you missed it Joe Donegan announced this at the last BioBeers and I could have broken the whole to wide open.

So that’s two News stories I scooped at BioBeers that I haven’t broken.  The other was, of course, the Bruce Ivins’ suicide.

Anyways, here’s a link and nice story from GlobeSt.com (I sworwe I had seen this story inthe Gazette or Fred News-Post, but can’t find the story in either of them):

Life Technologies has inked a lease to occupy an entire industrial/flex building here owned by J&N Properties. The 56,438-square-foot lease is a ten-year term for the biotech company, which also plans to expand the building for pharmaceutical distribution, according to Chad Tyler of Tyler Donegan Real Estate. Tyler, along with Joseph Donegan represented the landlord in the transaction. CBRE’s David Palank and Frank Graybeal repped the tenant.
The building, located at 7311 Governor’s Way, had been vacant, Tyler tells GlobeSt.com. “Life Technologies will be spending about $1.5 million to renovate it,” he says. The asking rate for the building had been $7.25 per square foot, triple net.

From what I heard, the plans are to build out the former PGC Scientific space into clean rooms and state of the art distribution facilitys for cGMP production of therapeutic (presumably cell-based) products.

If this is true, then Frederick County will likely be the largest producer of mammailan cell culture therapeutic productss on the Earth, combined with the Lonza-Walkersville site.

And a really interesting story in the Washington post about how the DC area is the Center of the Stem Cell Universe, or at least the most desirable

College Graduates Flock to DC Area

The Associated Press: Tuesday, May 26, 2009; 7:21 AM

“The Washington area is attracting recent college graduates because the chances of landing a job remain high despite the economic downturn, experts said.
The area is “adding jobs in [service-providing industries] and in health care and in the federal sector,” said George Mason University economist Stephen Fuller. “And the jobs we’re losing in the region,” such as construction and retail sales, “tend not to be college graduate-type jobs.”

FedBizOps Opportunities at Ft Detrick: Where’s the Beef?

I was stunned last week when I talking with an acquaintance of mine who is a Contract Specialist at SAIC-Frederick.  I was stunned because I didn’t know he was in the Science business (technically, he’s more like a purchasing agent) and because he was complaining that he gets so little responses from local suppliers RFI and RFQ that many times these “Purchase Orders” just expire.  I told him that I’d post some of these current opportunities, because I know many FredCoBio companies would love to have the chance to get a contract through SAIC.

Here’s the Beef.  A full  palette of delicious, juicy contracts just waiting to be bid upon. Viewable on FedBizOps:

S09-112 – Cell Line Development Services If no one jumps on this one, I’ll scream!!

Response Date: May 1, 2009 by 4:00PM Eastern

Archive date: May 30, 2009

Classification Code: A – Research and Development NAICS: 541711

Set Aside: N/A

Contracting office address: SAIC-Frederick, Inc.
National Cancer Institute at Frederick
92 Thomas Johnson Drive, Suite 250
Frederick, MD 21702-1201

Summary:

A contract manufacturing organization or supplier with the ability to economically develop a mammalian cell line expressing a therapeutic protein including but not limited to chimeric or humanized monoclonal antibodies, antibody fragment fusion proteins, or recombinant cytokines in an established cell line, such as CHO suspension cells, HEK 293 cells, or NS0 cells with stable expression level at 10-50pg/cell/day using a batch, fed-batch, or perfusion process.

Service Requirement:

Potential respondents are to provide detailed description of technical approaches and experience, as well as estimated costs in performing the activities detailed below:

Product Gene

1) SAIC-F would provide a gene sequence of an antibody or antibody fragment fusion with a protein, or a recombinant protein with construct size <10-12KB.
2) Respondents will provide description of sequence analysis and codons optimization for expression in mammalian cells using either direct synthesis or codon optimization techniques.

Product Expression Regulation

1) Respondents will provide a description of promoter; e.g., CMV or equivalent strong mammalian cell promoters.
2) Respondents will use a leading peptide to express the protein as secreted form.
3) Respondents will use a terminator or terminators to enhance target gene transcription.
4) Respondents will provide details on removal of introns to improve transcriptional yield where acceptable.

Cloning Requirement

1) Respondents will describe a selection system (GS or dhfr-) to be used in the expression system. Alternatives to the use of a selection system may also be described by the respondent.
2) Respondents will describe an additional selection to facilitate enriching high producer may be described; however SAIC-F requires to use a system previously approved by FDA for clinical trials.
3) Full length plasmid sequencing is required. A high transgene copy number in the cell line is preferred but not required.

Cell Line Requirement

1) Respondents will describe the proposed host cell line, such as CHO suspension cells, HEK 293 cells, or NS0 cells.
2) Respondents will describe how the host cell lines are qualified for use.
3) Respondents will provide how sequencing of the promoter and transgene coding region in the selected high producer is performed. Messenger RNA sequence and information regarding the location of the transgene(s) in the host cell is preferred but not required.

Procedure Requirement

1) Respondents will describe procedures for how the constructed plasmid DNA is transfected into a selected cell line; to include:
a) A stable transfection pool.
b) Single cell cloning methods.
2) If a selection system is used in expression, a reasonable transfected clone number (depend upon the system) is required; please describe. If there is no selection system is used to enrich high producer, more than 1000 single clones are required to select for expression screening.
3) Responder will describe procedures to evaluate cell growth and productivity based on respondent’s selection on medium and culture condition.
Cell Growth and Productivity Requirement

1) Respondents will describe how the following that would be delivered:
a) A serum-free or protein-free medium adaptation.
b) Cell growth to reach 5-10 a 106 cells/mL.
c) Cell doubling time in the range of 20-30hour.
d) Viability of culture greater than 90%.
e) More than 5 days for batch process or more than 9 days for fed-batch process.
f) Productivity of greater than 10pg/cell/day is required.
2) Respondents will describe methods used to characterize the expression candidate protein or previous experience working with clients to achieve characterization.

Key Requirements to be considered for the areas above include:

1) A traceable history of the host cell used in the study.
2) A clear history of the plasmid vector used in the study.
3) A cell culture medium that is GMP compliant.
4) Generation of a cell line expressing a product is well documented.
5) All the sequence information of the plasmid and cell line to be made available in standard electronic formats.

S09-135  Endotoxin Production

Purpose of RFI:

SAIC-F is to seeking to identify a qualified contract research organization that is able to economically produce a new batch of clinical grade bulk endotoxin that is compliant with current Good Manufacturing Practices to replace the > 30 year old bulk endotoxin currently in storage. PLEASE NOTE: An appropriate strain of E. coli would be required to be acquired by any potential Subcontractor should a Request for Proposal (RFP) and subsequent Subcontracting Agreement be offered to a selected offeror.

Project Goal:

The goal included in this statement of work is to produce a new batch of clinical grade bulk endotoxin that is compliant with current Good Manufacturing Practices to replace the > 30 year old bulk endotoxin currently in storage. This project will be broken down into a Development Section, including MCB production, and purification process development. An engineering run of bulk endotoxin along with a pilot lyophilization study. The final stage includes cGMP Production; including GMP bulk endotoxin manufacture and GMP vialing and lyophilization plus a manufacturing report.

Anticipated Milestones and Deliverables:

Milestone 1 – Activity A: Production and characterization of a 200 vial Master Cell Bank.

The previous cGMP endotoxin was produced from the Escherichia coli (Braude strain) group O 113:H10:K negative strain that was isolated and characterized at the Bacteriology Division, Bureau of Laboratories, Center for Disease Control, Atlanta, Georgia (Ewing WH, Hucks MC, and Taylor MW (1952) J Bacteriology 63: 319-325). The BDP will contact Dr. Epstein to determine if this bacterial strain is available from the NIH Clinical Center, the FDA, or the CDC.

The potential Subcontractor will obtain the appropriate strain of E. coli and then manufacture and characterize the 200 vial MCB according to current standards. The potential Subcontractor will provide a list of tests to be performed. SAIC-F may provide the potential Subcontractor a source of the required strain if necessary.

Milestone 1 – Activity B: Purification Development.

R&D grade E. coli cultures will be produced using the previously recommended chemically defined media and conditions. These cultures will be produced in the potential Subcontractor’s laboratories using standard R&D procedures and it is expected that this process will require minimal development work.

The resulting cell pellets will be used for purification development following the previous purification process used > 30 years ago. The potential Subcontractor will provide a proposal for process modification (if any) to meet the required scale, cGMP compliance, and product quality. Such studies may include:

1. A 10 liter culture will be produced and subjected to phenol extraction to produce a single unpurified bulk that can be used for purification development studies.
2. Tests to replace a dialysis step with transmembrane flow filtration (TFF).
3. Determine whether the deoxyribonuclease digestion can be replaced with Benzonase digestion.
4. Testing chromatography as a replacement for the sodium acetate/ethanol concentration step.
5. Determine whether the final dialysis step can be replaced with TFF.

Milestone 1 – Activity C: R&D Assay Development.

The potential Subcontractor will identify any method development necessary to perform the required release testing for the bulk and final vialed product shown in Tables 1 and 2.

SPECIAL NOTE: SAIC-F, with the NCI, will contact the US FDA for review of the proposed methods of analysis and specifications prior to initiation of the cGMP phase of the program. The results of that future discussion will be shared with the potential Subcontractor.

Milestone 2: Engineering Run/Pilot Lyophilization

The potential Subcontractor’s laboratories will be used to perform a cGMP pilot run. A full scale production and purification will be performed to test the process and produce bulk drug substance. This drug substance will be used as an internal reference standard for the following studies:

1. QC assay development.
2. QC testing for comparability to previous endotoxin CC-RE-Lot3.
3. Drug Substance stability studies.

The potential Subcontractor may perform any or all of these studies. The potential Subcontractor will also perform pilot formulation and lyophilization studies based on requirements to produce final vialed product listed in Table 3.

Milestones 3 &4: Production of cGMP Endotoxin

The potential Subcontractor will perform a full scale production and purification to produce cGMP grade endotoxin including the following activities:

1. Controlled fermentation will be conducted in a batch mode.
2. Purification will be performed to produce bulk drug substance.
3. QC/QA release of the drug substance.
4. Formulation, vialing, and labeling of endotoxin drug product (FVP).
5. QC/QA release of the FVP.
6. Stability program for FVP and, if required, bulk biological substance
7. Preparation of a Manufacturing Report (Appendix 1) to be included in
submissions to the US FDA with authorization to cross-reference any required
potential Subcontractor submissions to enable full review of the manufacturing
and testing documents by the US FDA for IND studies.

Table 1: Methods of Analysis and Specification for Bulk Biological Substance

Method Product Specification
Kinetic – QCL Potency Assay (EU/mL) (3) NLT 50,000 EU/mL
Gel Clot Potency Assay (EU/mL) (3) NLT 50,000 EU/mL
Sterility(1) Negative per 21CFR610.12
Residual host cell protein For Information Only
Residual host DNA For Information Only
Residual phenol(2) For Information Only
Mass spectrometry; method to be proposed by Contractor For Information Only
Polysaccharide characterization; method to be proposed by Contractor For Information Only
(1) Bacteriostasis & fungistatsis analysis to be performed on representative sample
(2) Additional residuals analysis may be required based on the potential process-related contaminants.
(3) Stability indicating assay

Table 2: Methods of Analysis and Specification for Final Vialed Product

Method Product Specification
Kinetic – QCL Potency Assay (EU/vial) 5,000 – 15,000 EU
Gel Clot Potency Assay (EU/vial) 5,000 – 15,000 EU
Sterility Negative per 21CFR610.12 & cUSP
Residual moisture <=3.0%
Note: All methods are stability indicating assays

Table 3: Required Deliverables for Milestone 3 & Milestone 4

CGMP Production Stage Minimum Quantity Configuration
Final Bulk Biological Substance 1.25 x 108 EU from 62.5 liters Final buffer is 1% lactose, 0.1% PEG-6000 to be bulk filled into sterile glass 10L bottles (8 L per bottle) and stored at 2-8oC
Final Vialed Product, Lyophilized 10,000 vials, net stability and release sampling; 2,000 EU/mL when reconstituted with 5 mL Water-For-Injection (WFI) 10,000 EU per vial in Type II borosilicate glass vials with 20mm stoppers and 20 mm West Aluminum seals. Sterile, single use vials stored at 2-8oC

S09-117 – DNA Plasmid Development Services

Summary:

A contract manufacturing organization or supplier with the ability to develop a process, and perform manufacturing for DNA plasmids for therapeutics and vaccines, using an established cell line, such as E. coli DH 5 alpha with productivity of not less than 800mg/liter of purified plasmid DNA in fermentation broth. It is also required to demonstrate that high quality of plasmid product including but not limited greater than 90% of supercoiled DNA can be purified from the broth.

Service Requirement:

Potential Offerors are to provide detailed description of technical approaches and experience, as well as estimated costs in performing the activities detailed below:

Standard Plasmid Construct – Assume a gene insert would be provided to the Offeror for the purposes of responding to this Request for Information/Sources-Sought Notice:

1) Plasmid contains several basic components including but not limited to promoter(s) to allow gene expression in a mammalian cells and/or tissues; with bacterial replication capability in either high or low copy number, and with no beta-lactmase resistance marker.

2) Plasmid with size of less than 12Kb.

3) Plasmid carries at least one protein coding sequence for expression in mammalian cells and/or tissues.

Production System and Productivity

1) E. coli DH 5 alpha or equivalent FDA accepted E. coli strain that can be employed as host cell for production of a plasmid.

2) The E. coli cells used for production meet FDA requirements for GMP manufacturing. The Offeror is to provide standard listing of procedures to establish and characterize Master Cell Bank.

3) It is required to demonstrate that the productivity is 800mg/liter or higher of fermentation broth at 10 liter or larger fermentation scale. The Offeror is requested to provide experience with specific data.

4) It is required to demonstrate at small scale that the plasmid can be purified from the fermentation broth at reasonable purification yield. The Offeror is requested to provide experience with specific data.

Product Characterization and Quality Requirements

1) A full length plasmid DNA sequence is required; the Offeror is to provide methods used and identify any subcontract relationships, if applicable

2) It is required to demonstrate that the purified plasmid is acceptable in quality including but not limited to higher than 90% of supercoiled plasmid.

cGMP Production

1) The Offeror is to provide facility and operational description for production of purified plasmid DNA under GLP and/or cGMP conditions. Examples of previous experience are requested.

2) It is requested that production be demonstrated for at least 5 g purified plasmid DNA per batch. The Offeror is requested to provide information on experience for large scale DNA plasmid production.

Nature Methods a la Frederick

At the last BioBeers, I didn’t have a lot of time to talk to Jim Hartley.  He’s now with SAIC/NCI-Frederick working in the Protein Expression group with Deb Chatterjee.  It did give me great pleasure, though, as I was scrambling to get my laptop to communicate with the PC projector, to eavesdrop on a conversation Jim was having with Mike Smith about this new cell-free system they’ve come up with for screening genome wide protein expression.  An “all DNA” protein microarray, of sorts.  This was described in a September PLoS publication:

Protein Microarray On-Demand: A Novel Protein Microarray System

Deb K. Chatterjee^1,3*, Kalavathy Sitaraman^1,3#, Cassio Baptista^2,3#, James Hartley^1,3, Thomas M. Hill⁴, David J. Munroe³

1 Protein Expression Laboratory, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland, United States of America, 2 Laboratory of Molecular Technology, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland, United States of America, 3 Advanced Technology Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland, United States of America, 4 Department of Microbiology and Immunology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota, United States of America

Abstract

We describe a novel, simple and low-cost protein microarray strategy wherein the microarrays are generated by printing expression ready plasmid DNAs onto slides that can be converted into protein arrays on-demand. The printed expression plasmids serve dual purposes as they not only direct the synthesis of the protein of interest; they also serve to capture the newly synthesized proteins through a high affinity DNA-protein interaction. To accomplish this we have exploited the high-affinity binding (~3-7×10 ^-13 M) of E. coli Tus protein to Ter, a 20 bp DNA sequence involved in the regulation of E. coli DNA replication. In our system, each protein of interest is synthesized as a Tus fusion protein and each expression construct directing the protein synthesis contains embedded Ter DNA sequence. The embedded Ter sequence functions as a capture reagent for the newly synthesized Tus fusion protein. This “all DNA” microarray can be converted to a protein microarray on-demand without need for any additional capture reagent..

To take it back a step further, I saw Jim in a professional capacity at LTI frequently primarily because our names are nearly homophones (Hardy vs Hartley).  Seems like we were always swapping mail or phone messages (back in the days before e-mail).  And just to let you know, back in thise days Jim was working in R&D “cloning” the native restriction enzymes everyone uses today (which some bone-headed marketing guy dcided he’d share with NEB, but that’s a whole other story), working on recombinant TdT (a real popular enzyme back in the day we were making from frozen calf thyroids we bought from a meat packing plant.  Talk about variable yield..).  He may be best know for working on the team that discovered/commercialized the Gateway Cloning system.  he is also the inspiration for me starting BioBeers, and this is the first one he’s made it to.

Mike Smith worked on creating all the Competent Cells at LTI. Things like DH5 alpha, Electromax, LE cells. When IVGN laid us all off (yet another example of wasting talent, but don’t get me going again), he started GeneChoice (now a shell of its former self after being peddled around like rubbish by another bunch of marketing geniuses), making better comp cells than the one’s he created 10 years earlier.

Anyway, back to my original story.  Just reading the abstract in Nature Methods this week (you have to subscribe to Nature our buy the article on-line, despite this research being funded by our tax dollars on a Federally owned land).  The abstract is pretty sparse, but here it is (proper citation: Nature Methods 5, 1001 – 1002 (2008) doi:10.1038/nmeth1208-1001):

Comprehensive sets of clones and improved high-throughput methods for production of functional proteins now allow proteome-scale in vitro experiments on nearly 15,000 human genes

And lastly comes this article I just found from BioTechniques in September:
Biotechniques. 2008 Sep;45(3):307-15.

Identification of highly expressed, soluble proteins using an improved, high-throughput pooled ORF expression technology.

Waybright T, Gillette W, Esposito D, Stephens R, Lucas D, Hartley J, Veenstra T.Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC-Frederick, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
This article describes an improved pooled open reading frame (ORF) expression technology (POET) that uses recombinational cloning and solution-based tandem mass spectrometry (MS/MS) to identify ORFs that yield high levels of soluble, purified protein when expressed in Escherichia coli. Using this method, three identical pools of 512 human ORFs were subcloned, purified, and transfected into three separate E. coli cultures. After bulk expression and purification, the proteins from the three separate pools were digested into tryptic peptides. Each of these samples was subsequently analyzed in triplicate using reversed-phase high-performance liquid chromatography (LC) coupled directly online with MS/MS. The abundance of each protein was determined by calculating the average exponentially modified protein abundance index (emPAI) of each protein across the three protein pools. Human proteins that consistently gave high emPAI values were subjected to small-scale expression and purification. These clones showed high levels of expression of soluble protein. Conversely, proteins that were not observed by LC-MS/MS did not show any detectable soluble expression in small-scale validation studies. Using this improved POET method allows the expression characteristics of hundreds of proteins to be quickly determined in a single experiment.