I want to make pellets consisting of mainly the following:

  • microcrystalline cellulose
  • saccharose
  • rice starch
  • ascorbic acid
  • glycerin

and an enzyme as active component.

Is it furthermore necessary to add protease inhibitors or other stabilizers to remain the activity of the enzymes for a periode of at least one year OR is the dry environment conservation enough?

The enzyme is called histaminase and is extracted from kidney cortex through a series of centrifugation, filtration and dialysation. To be more precise, it is a liquid protein extract containing small amounts of this enzyme. No buffer etc. is added to the protein extract so far. The whole purification process is done at 4°C. I don't know how long the enzyme is stable without further preparation. Probably not that long because some proteases should remain in the natural homogenate from porcine kidney cortex. The other components of the pellets are of pharmaceutical quality. The aim of this experiment is to feed the pellets to dogs with digestive complaints. For this purpose the pellets will get a enteric coating to survive acid environment of the stomach.

  • $\begingroup$ So you're trying to make pills? $\endgroup$ – MattDMo Aug 20 '16 at 16:23
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    $\begingroup$ Other questions: what is the enzyme? How is it purified, and how pure is it? Has it been lyophilized? What storage condition requirements do you have - room temp? +4? -20? -80? What is the stability of your prepared enzyme, without putting it into pill form? Does it have BSA or other carrier proteins with it? If so, at what concentration? Are your other materials sterile? Have they been tested for protein content? As you can see, a lot of factors go into determining the stability of a protein. Please use the edit link to add all of the requested information into your question. $\endgroup$ – MattDMo Aug 20 '16 at 16:31
  • $\begingroup$ @MattDMo hope the added informations help $\endgroup$ – Luke Aug 21 '16 at 13:57

As you can probably guess from my comment, there are a lot of factors to take into account when thinking about protein stability. Here's what I would recommend you do.

First, make sure your extract is in a neutral buffer, like PBS, HEPES, or something similar. This is a fairly standard procedure when working with proteins. Next, since this is a kidney extract, there are likely to be gobs of proteases around, so I would strongly recommend adding broad-spectrum protease inhibitors, and keeping the extract as cold as possible to inhibit any enzyme activity. I would also add BSA at 10 mg/ml, both to stabilize the proteins and keep them from precipitating while in solution, and to lessen the chances that any proteases present will attack your enzyme of interest.

Once you have your solution stabilized, I would aliquot it and lyophilize it. Proteases are most active in solution, so once dried you'll reduce their activity even more. You can now add the inert ingredients (microcrystalline cellulose, etc.), mix thoroughly, press your tablets, and add the enteric coating. Finally, once everything is prepared, I would store the final product at as cold a temperature as is convenient. Again, this will inhibit any enzyme activity and prevent degradation.

I don't know if you have a functional assay to determine activity of the enzyme of interest, but if you don't I'd highly recommend developing one if at all possible. This way, you can test the activity of the fresh preparation of extract and compare it to various stages of processing into tablets, especially the finished goods, to ensure that you haven't lost much activity. I would also test random tablets throughout the year to make sure they haven't lost activity in storage, otherwise you won't be able to compare your experimental results.

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  • $\begingroup$ I don't want to press tablets. I plan to use pellets like those ones. So I don't have to lyophilize the extract because the process works with a moist compound. But I asked myself if the dogs could get in troubles because of bacteria etc. I centrifugalize at max. 50000g. So cells shouldn't remain in the liquid but what's with viruses?! $\endgroup$ – Luke Aug 21 '16 at 16:03
  • $\begingroup$ Furthermore I read that most of the proteases like PMSF or AEBSF are toxic for cells. So is there a solution for the case of feeding dogs with the pellets? $\endgroup$ – Luke Aug 21 '16 at 16:38
  • $\begingroup$ @Luke I apologize, I misunderstood the type of pill you were making. The reason I suggested lyophilization was not to just dry out your extract, but to preserve it and help protect it against proteases and other degradation, so even through you're making capsules, I'd try to keep everything as dry as possible. As far as the bacteria/virus question, you'll have to decide that for yourself. We (and dogs) don't eat sterile food, everything has various microorganisms in it. Dogs are able to tolerate a wide variety of them, what with the anal licking and eating food off the ground and whatnot. $\endgroup$ – MattDMo Aug 22 '16 at 14:45
  • $\begingroup$ @Luke as with all potential toxins, the dose makes the poison. Figure out how much of those inhibitors you'd like to add, as well as compounds like leupeptin and aprotinin, look up their LD50 or toxic dose, then calculate out milligrams of inhibitor (or micrograms, whatever) per kilogram of canine, and consult with a toxicologist if necessary. Search around, you may be able to find safer inhibitors. However, throughout all of this, an enzymatic functional assay is vitally important for making decisions about dose and stability - it may turn out you need few or no inhibitors at all. $\endgroup$ – MattDMo Aug 22 '16 at 14:49
  • $\begingroup$ I know this question was some while ago, but maybe you could help me out again ;-) After the centrifugation I want to make a ammoniumsulfate precipitation with the supernant. Do you think that I have to do a dialysis to remove the ammoniumsulfate OR could I use the homogenate direct without any further preparation in my pellets? $\endgroup$ – Luke Oct 28 '16 at 9:56

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