I have been asked how I prepared GDF11 powder for injection in tiny picogram doses, so I am going to detail this here. The trick with picogram dosing is serial dilution of the GDF11 protein using two bottles of bacteriostatic water, as explained below.
What You Will Need
Items you need to prepare GDF11 for injection:
- Two 30 ml bottles of Hospira bacteriostatic water
- A box of 1 ml sterile insulin syringes
- A box of fine 30 gauge (0.3 mm) sterile hypodermic needles
Bottle of GDF11 powder, two 30 ml bacteriostatic water bottles, 1 ml syringe, 30 gauge needles
You need 1 ml syringes graduated into a hundred 0.01 ml steps, as shown in the above picture, because we will be measuring out small 0.1 ml amounts of liquid. Such 1 ml syringes are called insulin syringes. In the UK you can buy these 1 ml syringes
here. In the US search
here.
I usually use Luer slip syringes and hypodermic needles (a system where needles push onto the syringes), rather than Luer-Lok (where needles lock on).
I use very fine 30 gauge hypodermic needles which are only 0.3 mm thick, because I am not a fan of large fat needles! You can buy 30 gauge sterile needles
here in the UK. In the US search
here. If you are using Luer slip syringes, make sure you get the corresponding Luer slip needles to go with them.
Hospira bacteriostatic water you can buy
here in the UK (it's expensive in the UK, at £15 a bottle), and
here in the US. You can get plain bacteriostatic water or sodium chloride bacteriostatic water; either is fine. You need two 30 ml bottles of bacteriostatic water. Hospira is the high quality brand of bacteriostatic water.
The Procedure for Preparing GDF11 for Injection
The 10 micrograms of GDF11 from Buckylabs arrives in a small sterile glass bottle, containing a tiny white speck of powder no larger than a sugar grain.
Snap off the plastic top protectors (shown in green in the above picture) from the two bottles of bacteriostatic water, and break off the plastic top protector from the Buckylabs bottle. This will reveal a gray rubber area in the center of a metal cap. Note that these bottles are never opened: liquid is introduced or removed from them by inserting a hypodermic needle through the rubber.
You want to clearly label one of your bacteriostatic water bottles as the MASTER, and the other bacteriostatic water bottle as the SECONDARY.
Then take a sterile needle and place it onto a syringe. Now push the needle through the rubber top of the MASTER bacteriostatic water bottle, and draw out approximately 1 ml of water into the syringe. Pull out the needle from the MASTER bottle, and then push this needle through the rubber top of the Bucklabs bottle, and inject in that 1 ml of water into the Bucklabs bottle.
Withdraw the needle, and give the Bucklabs bottle a vigorous shake for about a minute, to make sure all the GDF11 protein in the tiny white speck dissolves in the water. When the speck can no longer be seen, then all the GDF11 will be dissolved in the water.
Now re-insert the needle back into the Bucklabs bottle, and draw out every last drop of the water into the syringe, and re-inject that syringe full of water back into the MASTER bacteriostatic water bottle.
Once you have done this, without pulling out the needle, give the MASTER bottle a brief shake, and then draw out about 1 ml of water from the MASTER bottle into the syringe, and then immediately re-inject that water back into the MASTER bottle; this is done in order to wash out the needle and syringe of any high concentration liquid. Repeat this wash out step a few times. Then withdraw the needle.
Give the MASTER bottle a good shake for 15 seconds to mix it thoroughly.
So now in the MASTER bottle you will have 10 micrograms (10,000 nanograms) of GDF11 dissolved in 30 ml of bacteriostatic water. Which means that in 1 ml of water from the MASTER bottle, you will have
10000 / 30 = 333.3 nanograms of GDF11. And in each 0.1 ml, you will have 33.3 nanograms of GDF11.
Right, so now we are going to draw out some liquid from the MASTER bottle, and inject a precise 0.1 ml amount into the SECONDARY bottle of bacteriostatic water. So insert the needle into the MASTER bottle, draw out approximately 0.2 ml, and withdraw the needle from the bottle.
With the syringe held vertically and the needle pointing upwards, flick the syringe sharply with your fingernail to get out any air bubbles from the liquid in the syringe. We are going to inject a precise 0.1 ml amount into the SECONDARY bottle, so we need to remove out any air bubbles, otherwise it will affect our measurement of the 0.1 ml amount.
With the syringe still held vertically, press in the plunger and squirt out some of the liquid from the end of the needle, until the rubber bottom of the plunger lines up with the 0.1 ml mark on the syringe. Thus now we have precisely 0.1 ml of liquid ready to inject into the SECONDARY bottle. So now you insert the needle into the SECONDARY bacteriostatic water bottle, and inject in the 0.1 ml of liquid. It is important that you inject in precisely 0.1 ml, no more or no less. Withdraw the needle and give the SECONDARY bottle a good shake for 15 seconds.
That 0.1 ml of liquid you injected will contain
33.3 nanograms of GDF11, so that means in the SECONDARY bottle we now have 33.3 / (30+0.1) = 1.1 ng per ml, and thus 0.11 ng of GDF11 per 0.1 ml.
So finally we end up with the SECONDARY bottle containing 0.11 ng of GDF11 per 0.1 ml of water. Or equivalently, we can say:
There are 110 picograms of GDF11 in each 0.1 ml of liquid drawn from the SECONDARY bottle
So if you are aiming for a GDF11 dose of say 220 picograms administered once every two days, you would draw out 0.2 ml from the SECONDARY bottle, and inject that subcutaneously every two days.
Storing Bacteriostatic Water Bottles in the Fridge
Bacteriostatic water bottles must be stored in the fridge, and only taken out when you want to perform an injection. Be very careful not to mix up the MASTER and SECONDARY bottles. When injecting, you only want to draw from the SECONDARY bottle. The concentration of GDF11 in the MASTER bottle is far too high for injection purposes. You might want to wrap your MASTER bottle with clingfilm or aluminum foil to prevent you accidentally mistaking it for the SECONDARY bottle when you are going to inject.
Once your SECONDARY bottle runs out, you can buy a fresh bottle of bacteriostatic water, and create a new SECONDARY bottle by again injecting 0.1 ml from the MASTER bottle, using the steps above. Each time you create a new SECONDARY bottle, you will only draw out a tiny amount from the MASTER, so the MASTER bottle should in principle last indefinitely.
However in reality, bacteriostatic water bottles are only supposed to last for 28 days once you have started using them, at least according to the manufacturer's instructions, and after that the levels of bacterial growth in the bottle may increase. Though I have heard that those injecting GDF11 keep their bacteriostatic water bottles for a year in the fridge before throwing them away. Whether it is safe to keep them that long, I am not sure. It might be possible to keep them longer than 28 days, but I am not so sure about a year.
If you are going to try to extent the lifespan of these bacteriostatic water bottles in the fridge, I would suggest placing them in the coldest part of the fridge, where the temperature is below 5°C (below 5°C bacteria grow much more slowly). Next to the cooling element at the back of the fridge is typically around 5°C. But I would try to make sure the liquid does not freeze, as perhaps this could be harmful to the GDF11 protein (you can freeze the dry lyophilized GDF11 protein, but once it is reconstituted by dissolving into water, my guess is that freezing the water might stress and damage the protein).
I bought a digital fridge thermometer with maximum and minimum temperature recording to ensure that the temp in the area I placed by bacteriostatic water bottles was below 5°C, but remained above 0°C.
Another way to help reduce the bacterial levels in the bottle is to disinfect the rubber stopper with alcohol just before you push the hypodermic needle through the top. That helps prevent bacteria entering the bottle via the needle. I always disinfect my rubber tops in this way before pushing in the needle.
Steve Perry also provides some GDF11 preparation instructions in
this document, but they are a bit complicated, so hopefully the instructions I provided above will be simpler.
Remember that GDF11 is not sold for human consumption, and clinical trials of GDF11 on humans are planned soon but have not yet taken place, so at this stage, if you want to experiment with this, you are doing so at your own risk.