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Raw Heparin Processing

Heparin is present in a variety of animal tissues.  The most common source for pharmaceutical use is pig intestines.
As reported by the Wall Street Journal's Heath Blog on February 21, 2008 , Making Heparin is a Dirty Job.
As this picture from the blog also shows, Chinese suppliers were not using stainless steel equipment.  From The Pump Handle, a blog for the public health crowd, we can read their blog from February 28, 2008, China, Pig Intestines, and the FDA.  Read the description of heparin production and compare it with the patents that follow.
The floors had large puddles and drainage channels; the workshops were dilapidated and unheated; and steam from the production process fogged up the windows and soaked the walls. There were large ovens to cook ingredients and halls lined with barrels to store enzymes, resins, intestines and wastewater.

In addition to porcine sources, some heparin is produced from bovine sources.  In Jordan, Hikma is producing heparin from bovine sources from Argentina.  This heparin is marketed in Jordan and Saudi Arabia, where Muslim preferences are for non-porcine sources.  Teva, a Israeli pharmaceutical producer of generic drugs, supplies kosher heparin to Israel.  It seems that bovine heparin is produced from the lungs of cattle.  This seems like a second niche for Iowa heparin production.  In Comparison of bovine and porcine heparin in heparin antibody formation after cardiac surgery, Francis and coworkers report that bovine heparin is more likely to cause heparin-PF4 antibody formation than porcine heparin.

There are better ways to protect the world's heparin supply:

Easy way of isolating heparin from mucosa tissue

Assignee: Akzo Nobel (more recently reassigned to Merck)
Inventors: François Egbert Abraham Van Houdenhoven, Adrianus Lambertus Maria Sanders, Petrus Johannes Josephus Van Zuthpen
SUMMARY OF THE INVENTION

The present invention provides a new and easy way of isolating heparin from mucosa tissue. It has been found that protein in an aqueous mixture comprising mammalian mucosa tissue can also be digested with a proteolytic enzyme at a temperature between 50-75° C. for up to approximately 6 hours. A simple "pre-hydrolysis" step at ambient temperature can optionally be included. This was shown to improve the efficiency of the proteolysis significantly. After the raise in temperature hydrolysis can be continued preferably in the presence of polyanion adsorbent material at a temperature below 50° C. The digestion can be carried out in containers with intermediate capacity.

In the detailed description, the authors note that an advantage of their system is that is can be done on a relatively small scale.  In the patent application, batch sizes varied from 3kg to 250 kg.  So this method is able to match the scale of operation to the output of a single slaughterhouse.  The authors note:

Usually heparin extraction plants are not located at a short distance from the slaughterhouses where the mucosa is collected and transportation over long distances is required. Especially in remote areas this results in a delivery of low quality material at the production plant and an increase of costs.

The logical limit to this is to process the heparin in the slaughterhouse.

The authors also note that the reaction vessel can be stainless steel.  The goal would be to have the heparin produced at a rate that matches the rate that hogs are slaughtered.  Since the processing time is 6-24 hours, the reaction vessels need a combined volume sufficient for the volume of pig intestines slaughtered in a single day.

The simple pre-hydroelytic step involves a commercially available alkaline protease named Maxatase.RTM, from Gist Brocades, now DSM.  The process also uses an anion exchange resin to help with the extraction of the heparin from the solution.  I assume that this resin is available from Akzo Nobel.  The heparin is separated from the solution by elution.  This suggests that the bottom of the 'brewing container' should have a  stopcock or ball valve on the bottom.

By a turn of events, François Van Houdenhoven now lives near Chicago and is a director at Price Waterhouse Cooper.

To maximize efficiency, the heating can be provided by immersion heaters, such as Watlow's FIRERODTM.  In a similar  idea, the tank could also include UV lamps in quartz tubes to sterilize the brewing heparin raw material.  I tend to equate the tank with a vacuum chamber, and the Kurt J Lesker Company recommends using UV to aid in degassing UHV chambers.  They do warn that UV radiation below 240 nm can create ozone, which is ok for sterilization, but can react with mucous membranes, such as intestines.  So any application of in-tank UV would require testing.  The Heparin adsorption spectra have peaks around 200nm and 260nm, close to the 254mm mercury spectra line that is used in most UV germicidal lamps.  The UV can pass through glass, so UV lamps like those in the Ultraviolet Air Probe Sanitizer, mounted above the tank, should be a reasonable option.

Method to produce a solid form of heparin

US Patent 6,933,373 issued on August 23, 2005

Assignee: Warner-Lambert Company (part of Pfizer)

Inventors: Ranganatha Raghavan, Jay Lee Jett


SUMMARY OF THE INVENTION

This invention provides a new method to process a solution containing heparin to a solid form of heparin with a drum dryer operated at atmospheric or vacuum pressure and a suitable drying temperature. Preferably, a double drum dryer is used. The invention is further disclosed in the attached claims.

The solution processed with the invention contains heparin as a main component. It may be a solution from a heparin purification or manufacturing process, an Q extraction solution of animal mucosa, tissues or organs with contaminants, or a solution with contaminants eluted from resins absorbed heparin. Such contaminants could be, for example, glycocosaminoglycans, proteins, bacteria, dermatan sulfate, pathogens etc.

By applying the method of this invention, the production of solid heparin from its solution uses one-step operation by means of a drum dryer instead of multiple steps in a conventional extraction, production or purification process. Specifically, this invention provides excellent advantages to eliminate the requirement to use a large amount of dangerous and expensive organic solvents, to reduce the process cycle times, to allow the possibility of a continuous drying process, to improve energy efficiency and to reduce the risks of personnel safety in handling organic solvents.

The drum drying can be done under vacuum in under a minute.  One of the key advantages of this device is the use of vacuum processing to reduce organic solvents.  This eliminates the use of dangerous organic solvents and results in an easily handled dry heparin that can be easily shipped to the manufacturer of the heparin.

Double drum dryers are produced by Buflovak, Ltd of Buffalo, New York.  These use a steam ejector pump, which is similar to an industrial version of the atomic beam sources that I used in my dissertation and postdoctoral positions.  The Heparin Sodium specification limit the residual solvents.  By using these drying methods instead of using solvents, we can achieve high scores on this test of purity.

It is our ethical and regulatory responsibility to monitor the safety of the medicines we develop from their first introduction in the research laboratory, throughout clinical testing, and for as long as they are prescribed to patients anywhere in the world.
This view is consistent with our goal for heparin production.  So it is hoped that Pfizer would see fit to license this technology for the safety of the world's medicine supply.  Pfizer is also a supplier of drugs for animal use.  So Pfizer would also gain by a move by hop producers to certify the health of their hogs.

Ultrasonic mixing/disintegration

At high power, ultrasound can mix and even disintegrate tissue, which could improve the speed of producing heparin by the method of Van Houdenhoven's patent.  Coupling that with a 'sloshing' motion from a Stewart platform, should speed the process significantly.
Hielsher Ultrasound Technology produces ultrasonic mixers that use piezoelectric elements that can generate ultrasound at powers up to 16 KW.  They probably have competitors, so more investigation may be in order.  This seems to be a novel idea in heparin processing.

Facility Cleaning (a variation on what mom's advice: clean with soap and disinfect with sunlight)

I expect that the heparin production facility should be held to cleanroom standards.  This may well be overkill, but we should maintain high standards for cleanliness and freedom from pathogens.

For equipment cleaning, I am interested in using high purity detergents, such as the decon products.  When I was at Cambridge, we compared decon 90 (for stainless steel) and neutricon (for aluminum) with the standard UHV cleaning practice of trichloroethane, acetone and methyl alcohol.  The detergents actually did a better job, leaving only a layer of water that was easily removed from our stainless steel equipment.  For cleaning lab equipment, there is also dri-decon in powder form.

In order to assure high quality, some form of purified water is needed.  Deionized water would seem to be a reasonable solution, being much less expensive than distilled water.  Neither deionizing nor distilling assures that the water is free of pathogens, so either chemical or UV treatment may be needed.  Res-Kem is one manufacturer with pharmaceutical grade water purification (among many).

 Hanovia UV is a UK company that specializes in this very technology for improved cleanroom air quality.  UV water disinfection is also well established for high clarity water (suspended particles can shield pathogens), so the UV irradiation should follow all filtering.  The EPA has published ULTRAVIOLET DISINFECTION GUIDANCE MANUAL FOR THE FINAL LONG TERM 2 ENHANCED SURFACE WATER TREATMENT RULE, which provides guidance in treating drinking water.  UV lamps could also be used to sterilize the cleanroom itself, but only when users are not present.  ASHRAE Journal, August 2008 contains Utraviolet germicidal irradiation: Current Best Practices, which notes that that most effective frequency is 254 nm, the frequency of mercury vapor lamps.

It is reasonable to expect that hog intestines (as with any mammal's intestines/lungs) can be host to a variety of pathogens.  One way to address this is with ultraviolet germicidal irradiation.  One challenge may be that heparin has a UV peak near 240 nm that is due to photofragmentation, see figure 2 in Wavelength-Tunable Ultraviolet Photodissociation (UVPD) of Heparin-Derived Disaccharides in a Linear Ion Trap.  This peak is fairly broad and overlaps the 254 nm radiation used for ultraviolet germicidal radiation.  The source of the radiation is usually a mercury vapor lamp without any phosphors.  So is may be that using UV radiation could degrade the heparin.  In this case, the UV from this source would only be useful for disinfecting equipment, but not the mucosa tissue.

water absorption spectra
From the
UVB Primer, published at Colorado State, we see that 305 nm UV is considered to be quite dangerous, so it may be that the 304 nm radiation from a helium discharge lamp can also be used for ultraviolet germicidal irradiation, but this frequency is not going to cause dissociation in heparin.  From Water Adsorption Spectra,  published by London Southbank University,   we see that water is particularly transparent around 300-500 nm. So 304 mn radiation seems promising.  The recently published 

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