Pyro_cat
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People With Diabetes Making Their Own Insulin
Biohackers With Diabetes Are Making Their Own Insulin
Diabetes is a punishingly expensive disease. In an Oakland warehouse, scientists are going DIY.
https://elemental.medium.com/biohackers-with-diabetes-are-ma...
In 1940, during World War II, she and her husband, Victor Saxl, fled to Shanghai, China. In Shanghai, a year later, Saxl was diagnosed with type 1
diabetes. When the Japanese attacked Pearl Harbor in 1941 the Japanese occupation of China was tightened, and soon all the pharmacies in Shanghai were
closed. Saxl had no legal access to insulin. It was possible to buy insulin on the black market using one-ounce gold bars for payment. But that was
not the safest option; one of Eva's friends died from using the black market insulin.
Eventually, Victor and Eva decided to get insulin another—highly unconventional—way: make it themselves. The book "Beckman's Internal
Medicine"[Note 1] described the methods that Dr. Frederick Banting and Charles Best first used to extract insulin from the pancreases of dogs, calves,
and cows in 1921.
https://en.wikipedia.org/wiki/Eva_Saxl
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Metacelsus
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I can make my own insulin, and I don't even have diabetes 
On a more serious note, I worry about safety for this. Injectable medications need to be sterile and free of bacterial toxins. And with insulin, the
dosage must be carefully controlled (an overdose is fatal). If the alternative is death, it's worth it. But I don't think these people are following
CGMP.
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Ubya
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how are they going to measure the dosage? every batch is going to be different, as metacelsus pointed out, overdosing is quite easy, you need very
little insulin. i really doubt they can produce cheaper insulin in a domestic setting, and of good enough quality. each batch needs to be tested,
tests can be expensive if not in house (if they want to test by themselves good luck paying for the equipment).
surely it can be done, but not for 20$ a vial, you would need economy of scales, and a small production can't lower the costs
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Pyro_cat
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2018 - Alec Raeshawn Smith was 23 when diagnosed with Type 1 diabetes and 26 when he died. He couldn't afford $1300 per month for his insulin ...
Approximately 7.4 million Americans with diabetes use one or more formulations of insulin.
My idea is for diabetics to get together and start making it instead. Essentially starting small pharmaceutical companies.
People are already building their own closed loop systems. #T1D #WeAreNotWaiting #Loop
https://twitter.com/hashtag/wearenotwaiting
DIY is where its at. Plenty of smart people.
How difficult is this process of making insulin ?
[Edited on 22-8-2019 by Pyro_cat]
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Ubya
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Synthesizing human insulin is a multi-step biochemical process that depends on basic recombinant DNA techniques and an understanding of the insulin
gene. DNA carries the instructions for how the body works and one small segment of the DNA, the insulin gene, codes for the protein insulin.
Manufacturers manipulate the biological precursor to insulin so that it grows inside simple bacteria. While manufacturers each have their own
variations, there are two basic methods to manufacture human insulin.
Working with human insulin
1 The insulin gene is a protein consisting of two separate chains of amino acids, an A above a B chain, that are held together with bonds. Amino
acids are the basic units that build all proteins. The insulin A chain consists of 21 amino acids and the B chain has 30.
2 Before becoming an active insulin protein, insulin is first produced as preproinsulin. This is one single long protein chain with the A and B
chains not yet separated, a section in the middle linking the chains together and a signal sequence at one end telling the protein when to start
secreting outside the cell. After preproinsulin, the chain evolves into proinsulin, still a single chain but without the signaling sequence. Then
comes the active protein insulin, the protein without the section linking the A and B chains. At each step, the protein needs specific enzymes
(proteins that carry out chemical reactions) to produce the next form of insulin.
STARTING WITH A AND B
3 One method of manufacturing insulin is to grow the two insulin chains separately. This will avoid manufacturing each of the specific enzymes
needed. Manufacturers need the two mini-genes: one that produces the A chain and one for the B chain. Since the exact DNA sequence of each chain is
known, they synthesize each mini-gene's DNA in an amino acid sequencing machine.
4 These two DNA molecules are then inserted into plasmids, small circular pieces of DNA that are more readily taken up by the host's DNA.
5 Manufacturers first insert the plasmids into a non-harmful type of the bacterium E. coli. They insert it next to the lacZ gene. LacZ encodes for
8-galactosidase, a gene widely used in recombinant DNA procedures because it is easy to find and cut, allowing the insulin to be readily removed so
that it does not get lost in the bacterium's DNA. Next to this gene is the amino acid methionine, which starts the protein formation.
6 The recombinant, newly formed, plasmids are mixed up with the bacterial cells. Plasmids enter the bacteria in a process called transfection.
Manufacturers can add to the cells DNA ligase, an enzyme that acts like glue to help the plasmid stick to the bacterium's DNA.
7 The bacteria synthesizing the insulin then undergo a fermentation process. They are grown at optimal temperatures in large tanks in
manufacturing plants. The millions of bacteria replicate roughly every 20 minutes through cell mitosis, and each expresses the insulin gene.
8 After multiplying, the cells are taken out of the tanks and broken open to extract the DNA. One common way this is done is by first adding a
mixture of lysozome that digest the outer layer of the cell wall, then adding a detergent mixture that separates the fatty cell wall membrane. The
bacterium's DNA is then treated with cyanogen bromide, a reagent that splits protein chains at the methionine residues. This separates the insulin
chains from the rest of the DNA.
9 The two chains are then mixed together and joined by disulfide bonds through the reduction-reoxidation reaction. An oxidizing agent (a material
that causes oxidization or the transfer of an electron) is added. The batch is then placed in a centrifuge, a mechanical device that spins quickly to
separate cell components by size and density.
10 The DNA mixture is then purified so that only the insulin chains remain. Manufacturers can purify the mixture through several chromatography,
or separation, techniques that exploit differences in the molecule's charge, size, and affinity to water. Procedures used include an ion-exchange
column, reverse-phase high performance liquid chromatography, and a gel filtration chromatography column. Manufacturers can test insulin batches to
ensure none of the bacteria's E. coli proteins are mixed in with the insulin. They use a marker protein that lets them detect E. coli DNA. They can
then determine that the purification process removes the E. coli bacteria.
PROINSULIN PROCESS
11 Starting in 1986, manufacturers began to use another method to synthesize human insulin. They started with the direct precursor to the insulin
gene, proinsulin. Many of the steps are the same as when producing insulin with the A and B chains, except in this method the amino acid machine
synthesizes the proinsulin gene.
12 The sequence that codes for proinsulin is inserted into the non-pathogenic E. coli bacteria. The bacteria go through the fermentation process
where it reproduces and produces proinsulin. Then the connecting sequence between the A and B chains is spliced away with an enzyme and the resulting
insulin is purified.
13 At the end of the manufacturing process ingredients are added to insulin to prevent bacteria and help maintain a neutral balance between acids
and bases. Ingredients are also added to intermediate and long-acting insulin to produce the desired duration type of insulin. This is the traditional
method of producing longer-acting insulin. Manufacturers add ingredients to the purified insulin that prolong their actions, such as zinc oxide. These
additives delay absorption in the body. Additives vary among different brands of the same type of insulin.
Quality Control
After synthesizing the human insulin, the structure and purity of the insulin batches are tested through several different methods. High performance
liquid chromatography is used to determine if there are any impurities in the insulin. Other separation techniques, such as X-ray crystallography, gel
filtration, and amino acid sequencing, are also performed. Manufacturers also test the vial's packaging to ensure it is sealed properly.
Manufacturing for human insulin must comply with National Institutes of Health procedures for large-scale operations. The United States Food and Drug
Administration must approve all manufactured insulin.
Read more: http://www.madehow.com/Volume-7/Insulin.html
not that easy
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Felis Corax
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Sorry Ubya, but that article was written by someone who doesn't know the difference between proteins (specifically folded molecular machines made of
one or more polypeptide) and DNA (chains of paired nucleotides). DNA codes for protein synthesis, along other things, but it's not itself a protein.
Think of it as the difference between a blueprint (DNA) and a machine built from that blueprint (a protein).
There are other mistakes too, but most could be attributed to oversimplification. Describing DNA as being made of amino acids is unambiguously and
fundamentally wrong.
I'm not saying diy insulin synthesis is safe and easy, I haven't researched it, but the article referenced is not a source for anything besides
dubious infotainment for those without any knowledge of molecular biology. Well, that and as a bracing reminder of the importance of using reliable
sources.
Nothing is pure, nothing is perfect, nothing is clean.
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Pyro_cat
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I just found this https://openinsulin.org/
So they call this biohacking.
I find so much more interesting search results when I click on images instead of "all". I was looking at some stuff and decides to search: Skids
& Units for Pharma Biotech Products "insulin" then clicked images to see what this stuff looks like and that is where I found the above link. The
regular search results are just generic mainstream news and websites.
I don't know why they have to call independent research "hacking" like they want to make it sound dirty or illegal or something.
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Sulaiman
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Maybe it would be safer and cheaper to go from the USA to Canada (or Cuba) ?
CAUTION : Hobby Chemist, not Professional or even Amateur
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Tsjerk
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Or come to the Netherlands.
Price of Fiasp10 ml 100 u/ml : 20 euros
USA price: 310 Dollars
We do pay the first 385 euros a year our self (in total for all care, not per medicine or treatment) and health insurance is mandatory, the cheapest
being around 120 euros a month. For those with a low income there is some free financing plan to cover this 120 euros though, which is maximally 99
euros a month for those earning less than 20500 euros a year, this goes to 0 about linearly until you earn about 30.000.
At least I now understand even better why there is huge trafficking between the USA and it's neighbors...
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Ubya
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Quote: Originally posted by Felis Corax  | Sorry Ubya, but that article was written by someone who doesn't know the difference between proteins (specifically folded molecular machines made of
one or more polypeptide) and DNA (chains of paired nucleotides). DNA codes for protein synthesis, along other things, but it's not itself a protein.
Think of it as the difference between a blueprint (DNA) and a machine built from that blueprint (a protein).
There are other mistakes too, but most could be attributed to oversimplification. Describing DNA as being made of amino acids is unambiguously and
fundamentally wrong.
I'm not saying diy insulin synthesis is safe and easy, I haven't researched it, but the article referenced is not a source for anything besides
dubious infotainment for those without any knowledge of molecular biology. Well, that and as a bracing reminder of the importance of using reliable
sources. |
yep i know, i read it before posting, there are many errors but it was good enough to give an idea of the process, not a detailed procedure
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Metacelsus
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Making insulin would be quite easy for anyone with basic molecular biology skills. As I mentioned before, the issue is purification and formulation.
This would require expensive equipment as well as technical expertise.
The openinsulin website doesn't have many details, so I can't really judge whether they're likely to be successful with this. But then again, absence
of important details might be a bad sign.
[Edited on 2019-8-23 by Metacelsus]
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Pyro_cat
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I am new diabetic. Always been a mad scientist.
Don't underestimate our resentments towards this system that uses our sickness to make us cash cows.
Not only cash cows but when its time to buy needles you are an addict till you prove otherwise. I just order online now.
Its actually quite sad. Why would anyone do all the school required to be a pharmacist just to be part of the state pill police ?? Take pills from big
bottle put in small bottle then paperwork bullshit. Its not like the old days, before my time, when they were actual chemists. I have no respect for
them.
This is international forum. As American I am ashamed. I am older and it wasn't always like this. Wasn't always a big brother rip you off police
state.
No more politics, exploring science is how I chill so I should leave that alone. This is a great idea micro breweries for insulin just like with
beer. I hope it catches on.
Anyway I posted this here to attract good "biohackers" to our plight.
[Edited on 25-9-2019 by Pyro_cat]
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rockyit98
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What I've Learned 1.15M subscribers watch this channel first
https://www.youtube.com/channel/UCqYPhGiB9tkShZorfgcL2lA/vid...
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Chalo
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DIY insulin
The rather lengthy exposition by Felis Corax on how molecular biologists construct a plasmid that causes its host bacterium to overexpress a peptide
such as insulin omits the most important aspect of this process, namely that it only needs to be done once. The plasmids, all of which were developed
decades ago, do not have to be reconstructed every time someone wants to brew up some insulin. Only the fermentation and purification need to be done
continuously. The development costs for recombinant insulin were paid off long ago, yet they charge MORE rather than less.
A DIY developer purifying a protein today would not use elaborate and expensive chromatography but instead use one step affinity tags. For big
companies that already have large-scale columns this is actually much cheaper, and sometimes difficult peptides require the old skills (insulins not
one of them) but grad students by the thousands are right now purifying recombinant proteins who started from nothing but a sequence a few weeks ago.
The best thing that the hacker insulin project in oakland could do is duplicate the first plasmid expressing natural insulin (patent ran out decades
ago) and make it open-source, distributed freely to all professional and amateur investigators dried on a blotter so it can be mailed in ordinary
envelope. They could also distribute transformed bacteria as stabs in glass vials to save people the transformation step. Fermentation is only
expensive and difficult if a pharmaceutical company does it; ask anyone who brews beer.
Quality control, and legal hassles have far more to do with the expense than any technical difficulty. No one would dare actually give homebrew
insulin to a human for fear of liability. The true value in this project is simply to prove that as far as actual production costs, it can be made for
less than a dollar a dose.
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Metallophile
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I've been on insulin for over 10 years now. I remember googling it back then, and being blown away by how complex this molecule is! Biochemistry is
just mind boggling. I could maybe see someone extracting something from an animal pancreas, but I don't see them making it synthetically. Either way,
you'd me nuts to inject yourself with this! I have enough trouble with hypoglycemia, even using the real stuff.
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pneumatician
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Mood: ■■■■■■■■■■ INRI ■■■■■■■■■■ ** Igne Natura Renovatur Integra **
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Recover your pancreas?
oh no, snake oil!!! but a lot of people say: near recovered my pancreas functionally at 80, 90%...
so if it where me, I check Stevia, how people in Nicaragua control their sugar level in blood & etc.
"Stevia rebaudiana bertoni" a plant used from ancient epochs by natives in America and "rediscovered" in modern times by a Catalan guy in Paraguai or
another country around...
So if I are traveling in a bad train... I jump from it at get another!!! I DON'T MERRY WITH ANYTHING, ANYBODY & ETC or you have your arse sold!!!
:-D
good luck!
https://www.youtube.com/watch?v=Rr8ljRgcJNM
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