Bruce Ivins Anthrax Vaccine Patent 6387665 shows couldn’t do it as FBI says

March 7, 2010

The FBI DOJ claim that Ivins produced the first batch of anthrax the weekend from Friday Sep 14 in the evening to Sunday evening Friday Sep 16 2001.  Ivins was in the lab 2 hours 15 minutes each night. Dxer at Case Closed Blog has found FBI 302’s that indicate this is the time it would take to check animals if Ivins was doing that work that weekend.  The second time period is from Friday Sep 28 2001 to Friday October 5 2001.  This period is when the FBI DOJ claim Ivins prepared the Senate anthrax letters in his lab at Ft. Detrick.  The following information shows that this would not be possible.  It would take 5 days using the single fermentor and that would produce too little anthrax.  The 5 days would require running during the week when others were present.  It would be a continuous 5 day run, not just at night for 5 days.  The following are Ivins own experiments using that fermentor reported in Ivins own patent application dated from 2000 before the 2001 time period.

Bruce Ivins et al applied for a patent in 2000 which was granted in 2002. This patent describes in detail the actual growing of anthrax by Bruce Ivins using the New Brunswick Bio-Flo 3000 that the FBI 302 report indicated was the fermentor at Ft. Detrick. The patent also describes use of the speed-vac. The patent gives a table with the yield in mg of protective antigen (PA) (not anthracis) using the 5 Liter fermentor after growth of several days.

http://www.freepatentsonline.com/6387665.html

“Fermentation conditions: The fermentations described here were carried out using a New Brunswick Bio-Flo 3000 equipped with a 5.0 liter working volume glass vessel and stainless steel headplate and hemispherical bottom cooling dish.”

“EXAMPLE 1

B. Anthracis ΔSterne-1(pPA102)CR4 was compared with its parent spore-forming strain B. anthracis ΔSterne-1(pPA102). Both organisms were plated onto sheep blood agar (a preferred medium for promoting bacterial spore production) and grown at 37° C. for 1 day, after which the temperature was lowered to 25° C. for 4 days. The two strains were also grown in liquid Leighton-Doi medium, which is designed to promote spore production, for 1 day at 37° C. followed by 4 days growth at 25° C. Growth from both agar and broth cultures were examined under phase contrast microscopy for the presence of spores. Growth from all four cultures were then resuspended in phosphate buffered saline to a concentration of about 10 9 colony-forming units (CFU) per ml. All four cultures were then heat shocked at 64° C. for 60 minutes to kill vegetative cells. Aliquots of 0.1 ml of the heat shocked material was then plated out onto sheep blood agar and incubated at 37° C. for 2 days. ”

“EXAMPLE 2

B. anthracis ΔSterne-1(pPA102)CR4 was grown in an FA medium fermentor culture. No spores were seen upon phase contract microscopic examination. Only medium-length and long chains of bacilli were seen. Dilution plate counts on the culture determined that the culture contained 1.86×10 9 CFU per ml. Three ml of culture was heat shocked at 60° C. for 60 minutes, then 0.2 ml was plated onto each of 5 plates of Tryptic soy agar. After incubation for 2 days at 37° C., no colonies were seen on the agar plates, indicating that spore production in the fermentor was less than 1 per 1.86×10 9 CFU. On two other fermentation runs with this strain, similar results were obtained. No revertants to the parent spore-forming phenotype were observed.

The above process using an FA medium fermentor culture was repeated using the parent strain B. anthracis ΔSterne-1(pPA102). Growth on the tryptic soy agar after heat shock resulted in a total of 1000 total colonies, indicating that the parent strain B . anthracis ΔSterne-1(pPA102) had about 1000 spores per ml in the FA medium, or 1 spore per 106 CFU in the non-heat shocked medium. ”

“TABLE 1
Summary of Aerobic ΔSterne-1(pPA102)CR4 Fermentations
Final
Final Final Yield Doubling
Conc. Yield (mg Specific Time
Fermentation (μg PA83/ (mg PA83/g Growth T D
Conditions ml PA83) DCW) Rate (min)
Aerobic, Batch 51 235 8.10 0.0132 min −1 53
Aerobic, Batch 64 301 10.7 0.0136 min −1 51
Aerobic, Batch 45 225 7.40 0.0136 min −1 51
pH constant
Aerobic, 68 360 ND 0.0116 min −1 60
Fed-Batch
(non-
continuous)
DCW = dry cell weight ”

The final protective antigen (PA)  yields ranged from 235mg to 360 mg. This was based on using the actual fermentor at Ft. Detrick in growth runs of 5 days as indicated in detail in the Example 1 quote.

You can search at the above link.

“For each O.D.600 determination, two appropriate dilutions were made and results were considered acceptable only when both dilutions yielded a linear response. DCWs were determined starting with a 2 hr point by centrifuging 10 mls of fermentation liquor at 11,953×g for 10 min, resuspending the cell pellet in 10 mls of sterile PBS and pelleting the cells again under the same conditions. The cell pellet was resuspended in a minimal volume of PBS and transferred quantitatively to a preweighted Eppendorf centrifuge tube and centrifuged at 14,000 rpm for 5 min. Excess PBS was removed and the cell pellet was dried in a speed-vac for 72 hrs under vacuum and a medium heat setting. A final analysis of the dry weight versus O.D. 600nm revealed that the relationship between the two parameters was adequately fit with a linear function. ”

The speed vac appears to be have been used for a small sample. This appears to be on a mLs sample, ie 10 milli liters.

This appears to settle it. Ivins could not produce the anthrax at Ft. Detrick using even the fermentor and the speed-vac. It would take 5 days.  (  The Senate anthrax contained 871mg per letter at least.  The yield of antigen is below this, but of anthrax spores might be higher than this amount.  Is it dried antigen?  A spore weighs less than a living cell so there is some offset depending on the weight of antigen in a living cell compared to how it was measured or estimated.)

The speed vac was used on a small sample of 10 milliliters. This is too small just as Ms. Ulrich indicated.

This patent was applied for in 2000. Ivins knew from this data that it would be impossible for him to produce the anthrax at his lab and convert it into high quality powder in the amounts in the Senate letters. The speed-vac could not process this volume. Ivins knew that. He is the principal person on the patent and is listed first out of alphabetical order. The patent also cites Ivins own papers.


Inventors:
Ivins, Bruce (Frederick, MD)
Worsham, Patricia (Jefferson, MD)
Friedlander, Arthur M. (Gaithersburg, MD)
Farchaus, Joseph W. (Frederick, MD)
Welkos, Susan L. (Frederick, MD) ”


Filing Date:
03/07/2000

“Method of making a vaccine for anthrax
United States Patent 6387665″

Search Detrick “bioflo 3000″

“Results 1 – 10 of about 56 for Detrick “bioflo 3000″. ”

This is further confirmation the New Brunswick 5-L Bioflo 3000 fermentor is the relevant fermentor at Ft. Detrick.

Search Bioflo 3000 Ivins

” Results 1 – 10 of about 20 for Bioflo 3000 Ivins.”

Mostly the patent but it appears another Ivins paper or two at least in the results.

This patent was previously discussed at Case Closed after Dxer posted an extract from it.

http://caseclosedbylewweinstein.wordpress.com/2009/09/25/nas-panel-meeting-92409-%E2%80%93-limited-information-available-since-the-nas-did-not-webcast-the-session-and-so-far-has-not-made-available-any-recording-of-the-session-or-powerpoints-of-the-pres/

The FBI 302 page 1 discusses the 5 Liter Bioflo 3000 as being at Ft. Detrick and then discusses a transfer to KBI.

http://foia.fbi.gov/amerithrax/847425.PDF

This is presumably the fermentor that was said by some to have been inoperative in fall 2001.

FBI misspells the name of the fermentor in the 302 report.

New Brunswick Scientific BioFlo III fermentor is correct name. Can search on Internet for it and find used models.

http://foia.fbi.gov/amerithrax/847425.PDF

See page 1 of 51 page pdf. FBI spells it:

“5 L Bio Flow III fermentor.”

Comment posted at

http://caseclosedbylewweinstein.wordpress.com/2010/03/06/anonymous-scientist-summarizes-the-fbis-case-against-dr-bruce-ivins-if-it-wasnt-so-serious-it-would-be-laughable/#comment-4355

==

You can see photos of the results of centrifugation by searching centrifuge pellet in Google and also in image search.

http://en.wikipedia.org/wiki/Centrifugation

“Centrifugation is a process that involves the use of the centrifugal force for the separation of mixtures, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis. Chemists and biologists may increase the effective gravitational force on a test tube so as to more rapidly and completely cause the precipitate (“pellet”) to gather on the bottom of the tube. The remaining solution is properly called the “supernate” or “supernatant liquid”. The supernatant liquid is then either quickly decanted from the tube without disturbing the precipitate, or withdrawn with a Pasteur pipette.”

You don’t end up with Senate letter quality dry non-clumping powder by just centrifuging. The pellet is still wet. Will dumping the pellet onto a plate and letting it air dry for 2 hours turn it into Senate letter quality powder?

Assuming the answer is no, is that why lyophilizers are used? But the lyophilizer was not available to Ivins and it would have required decontamination that would have been known to the lab. The FBI did not release any documents on the lyophilizer at Ft. Detrick? Nor mention it in the summary report that I recall. Nor documents on the speed vac such its size and capacity?

From page 19 of summary pdf

“Second, the evidence demonstrated that the perpetrator was familiar with key items of laboratory equipment used in microbiology research. All of the Ames anthrax existing in the 15
U.S. labs prior to the attacks was in liquid slurry form or on vegetative cell slants, rather than in powder form. Consequently, it was not possible for the perpetrator to merely steal an existing quantity of Ames spore powder “off the shelf,” because none was known to exist in the holdings of any laboratory. Even if the perpetrator stole a quantity of liquid Ames anthrax slurry, it would still have been necessary to dry the anthrax in order to produce a product like the one recovered from the envelopes. This drying procedure would have required either the type of laboratory equipment, such as a lyophilizer or speed-vac system, that was present in each of the 15 labs, or considerable time and space to air-dry. Alternatively, if the perpetrator stole only vegetative cells or a small quantity of spores to use as seed stock, not only would the perpetrator have to dry the anthrax, he would also have to subject the anthrax to two separate culturing and washing operations using an incubator and centrifuge.”

Footnote 17 on page 35 of summary

“Numerous microbiologists have concurred that two hours and 15 minutes would be enough time to dry Ba spores, depending on factors such as the quantity of starting material, the volume of liquid in which it was suspended, and whether a centrifuge was used to eliminate most of the water, leaving behind a pellet, or paste, capable of being dried in well under two hours.”

These two quotations from the summary don’t seem fully consistent on the feasibility of centrifuging and air drying in 2 hours the quantities involved in the mailings.

If you air dry the paste, you spread it out with your fingers? It doesn’t clump when it dries when you do this?

If you can air dry 2 grams of powdered anthrax in 2 hours to get Senate letter quality, why do they use lyophilizers overnight?

Results 1 – 100 of about 22,500 for “lyophilized overnight”.

Meaning air dry the paste after centrifuging.

“leaving behind a pellet, or paste, capable of being dried in well under two hours.”

Does that mean air dried or does that mean dried with a speed vac or dried with a lyophilizer in 2 hours? Can you dry 2 grams of anthrax powder in 2 hours with a lyophilizer? Or is that overnight? Or more?
What about with the speed vac at Ft. Detrick?

Are there logs and sign outs for these devices? Have they been released for this time period?

“She said it would take about an hour to dry one milliliter of wet anthrax spores in one vial in a SpeedVac. It would have been impossible for Ivins to have dried more than a liter, which would have been required for the amount of anthrax sent in the letters, in the time frame they were mailed, Ulrich said. ”


Ulrich was a principal investigator in the diagnostic systems division at USAMRIID.

http://www.herald-mail.com/?cmd=displaystory&story_id=200518&format=html


She said Ivins was upset the FBI was watching him, but handled it as well as he could. “I’ve never even seen him angry,” Ulrich said.

“Ulrich said she worked with Ivins at the U.S. Army Medical Research Institute of Infectious Diseases in Frederick, Md., for about six years. The person she knew doesn’t match the troubled past Ivins is alleged to have had, she said. ”

Results 1 – 10 of about 19,100 for speedvac bacillus mL.

mL 1 milliliters = 0.0338140227 US fluid ounces

Results 1 – 10 of about 3,950 for speedvac bacillus uL.

uL is microLiter or 1/1000 of a mL.

These are typical experimental sizes for bacillus using a speedvac.

==

http://caseclosedbylewweinstein.wordpress.com/2010/02/28/new-york-times-fbis-anthrax-case-against-dr-ivins-is-not-convincing-independent-assessment-is-needed/

http://foia.fbi.gov/amerithrax/847425.PDF

page 44 interview of Ivins.

“RMR-1029, which is a large preparation of Bacillus anthracis Ames spoers compiled from multiple productions grown in both, shaker flasks at USAMRIID and fermentors at Dugway Proving Ground.”

So the Ba at USAMRIID was grown in the shakers which from a prior page in this same document, page 26, were at different places and only one shaker in any one place. These were hard to find space in according to the interview on page 26. Thus the type of run to produce 22 liters of anthrax used to help make RMR-1029 would be difficult to do. It would require many days on one shaker, if there is one in BSL-3. Ivins could not simply have put 11 flasks large enough to hold 2 liters of liquid and simply let them run together.

Ivins estimated 20 liters to make 2 grams of anthrax. The two Senate letters were at least 2 *.871 grams if no allowance for wastage is made. So given the set up at USAMRIID, Ivins time of 2.15 hours a night in BSL-3 would not give him the ability to have the shaker space and time needed for the runs for either weekend.

The FBI should have asked these questions and more. Instead, when an interviewee raises these points, the FBI agent notes them down but doesn’t follow up with questions related to them.

It appears the FBI agents think its someone else’s responsibility to figure out how it was done, not theirs individually or collectively. This appears to be part of an attitude of its not their job to think about the technical stuff.

When its not the FBI’s job to think about the technical stuff, then they are reduced to the gossip. When this is not enough, they try to lean on the person to get them to break. This pattern was repeated with Hatfill and then Ivins.

The FBI did not start out in 2001 with the question of how was this produced? Dxer mentions no 302’s from 2002. Is that because they asked the questions then and were told it required days and weeks with a good lab?

http://foia.fbi.gov/amerithrax/847425.PDF

Page 42.

“Blank recalls Ba samples being kept in the walk-in freezer for very short periods of time surrounding experiments. However these samples were usually already diluted for aerosolization. Blank believes that all glass impingers containing Ba may have been stored in this walk-in, but only for very short periods. Blank does not recall storage of any larger stocks (e.g. 500 ml flasks) of Ba.”

Ivins patent anthracis 6387665

http://www.google.com/#hl=en&q=ivins+patent+anthracis+6387665&aq=f&aqi=&aql=&oq=&fp=3d121c88310e67e3

Download Patent at Google

http://www.google.com/patents?vid=USPAT6387665

speed vac “two hours” overnight

http://www.google.com/#hl=en&ei=-CWUS7uPM8qm8AbcjOWoBQ&sa=X&oi=spell&resnum=0&ct=result&cd=1&ved=0CAUQBSgA&q=speed+vac+%22two+hours%22+overnight&spell=1&fp=3d121c88310e67e3

http://www.fbi.gov/anthrax/amerithraxlinks.htm

Detrick “bioflo 3000”

http://www.google.com/search?hl=en&q=Detrick+%22bioflo+3000%22&aq=f&aqi=&aql=&oq=

Results 110 of about 56 for Detrick “bioflo 3000”

Melanie Ulrich comments on Ivins

http://www.herald-mail.com/?cmd=displaystory&story_id=200518&format=html&autoreload=true

==

Comment submitted at:

http://anthraxvaccine.blogspot.com/2010/03/my-summary-of-unresolved-issues-in.html

==

The yields stated in the Ivins patent are for the yield of PA not of the anthrax itself.  (Jim White pointed this out on his blog.)

“protective antigen (PA) ”

“The data presented in Table 1 demonstrated that the PA yield on a unit volume and biomass basis,”

Claim one of the patent states

“1. A method of making a vaccine comprising: incorporating a protective antigen produced by recombinant asporogenic B. anthracis with a pharmaceutically acceptable carrier, wherein said recombinant asporogenic B. anthracis was isolated from a ΔSterne-1(pPA102) strain of bacteria and said recombinant asporogenic B. anthracis does not have the ability to bind a dye when grown on Congo Red Agar.

So the yield of PA understates the yield of the anthrax used to grow it.

The time of 5 days is the same however for the antigen and the anthrax bacillus it is in.  If we assume the antigen mass is a fixed ratio to the anthrax cell mass it is in, and that each anthrax cell has the same mass, then production of antigen mass is proportional to the production of anthrax cells.

The number of anthrax cells grown depends on the time period they are grown.  The mass of antigen follows the same path in time.  So if it takes 5 days to grow the antigens, then it takes 5 days to grow the cells.

(We can relax the assumption of fixed ratios made originally by allowing for fixed over time distributions of the ratios or cell size.)

A spore weighs less than a living cell so there is some offset  depending on the weight of antigen in a living cell compared to how it was measured or estimated.  So the antigen mass is less than a vegetative cell that contains it.  However, the spore weighs less than the vegetative cell.  How the antigen mass is determined matters too.  Is it converted to a dry powder and what loss of weight does that entail compared to a vegetative cell becoming a spore and then weighed as a dry powder of spores?

=Correction comments posted at Meryl Nass and Case Closed.

Final version at Meryl Nass  of one comment:

We must distinguish the vegetative cell, the antigen and a spore made from a vegetative cell.  A spore weighs less than a vegetative cell so there is some offset  depending on the weight of antigen in a vegetative cell compared to how it was measured or estimated.

The antigen mass is less than a vegetative cell that contains it.  However, the spore weighs less than the vegetative cell.  How the antigen mass is determined matters too.  Is it converted to a dry powder and what loss of weight does that entail compared to a vegetative cell becoming a spore and then weighed as a dry powder of spores?

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One Response to “Bruce Ivins Anthrax Vaccine Patent 6387665 shows couldn’t do it as FBI says”

  1. Eric Nadler Says:

    Wow! Nicely done. The truth is out there. And this helps. Make sure you let us know if serious people question your argument.


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