White Papers
Peptide Purposes and Peculiarities
What is Water Activity?
Methylcobalamin and Light
Water, Water, Everywhere!
Why are T3/T4 Difficult to Make?
HCG Formulation Facts
Peptide Purposes and Peculiarities
Overview
The use of peptides in compounded preparations is gaining popularity, primarily due to their ability to treat a wide variety of maladies that often cannot be treated successfully any other way. Among these treatments are; anti-aging, body building, sexual disfunction, various cancers, bed wetting, macular degeneration, weight loss, promoting ovulation and fertility, wound healing, osteoarthritis, and much, much more. Most peptides have high specific activity, thus require very low amounts to accomplish their intended purpose. In many cases, these peptides are produced naturally in the bodies of humans and animals, but because they are present in such low amounts, they are seldom obtained and purified from these tissues. Therefore, it makes more since to make them synthetically.
While the process of synthesis can be straightforward, it is time-consuming and typically only produces a few grams at a time. Since peptides are made up of a specific sequence of amino acids, they are synthesized in a way that resembles box cars on a train, one amino acid added to the “train” at a time. Some contain only a handful of amino acids while others are more complex containing 40+ amino acids. Many are essentially linear trains with some folding, some are circular where the chain ends connect, while others are branched to form highly complex shapes with bends, helix coils, and disulfide bridging to maintain their structures. Some are even more complex where other moieties such as sugars, lipids, phosphates or acetates are also part of their structures.
Some peptides can be produced through genetic modification of bacteria, then purified through various processes to obtain an essentially pure peptide, while many others are produced by means of solid phase peptide synthesis (SPPS). During the SPPS process, the initial amino acid is attached to resin beads, then through a series of chemical synthesis cycles each successive amino acid is added one by one to complete the chain. Lastly, the now-finished peptide is cleaved from these resin beads by reaction with trifluoroacetic acid (TFA). Since there are several peptide chains being synthesized in the batch at the same time, there will be occasions in which one or more amino acids fails to couple as intended. These are called “failure sequences” and must be removed during a purification process. Some of these are so similar to the intended peptide that they can be very difficult to remove. To account for this, the resulting certificate of analysis (CoA) from the supplier will often report less than 100% purity even though the total reported peptide content can be 100%.
At Compounder’s International Analytical Laboratory (CIAL), we have tested the following peptide-containing formulations:
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AOD9604 – 16 amino acid peptide BPC-157: 15 amino acid peptide Bremelanotide (PT-141): 7 amino acid peptide CJC-1295 (GRF 1-29): 29 amino acid peptide Corticotropin (ACTH 1-39): 22 amino acid peptide Cosyntropin (ACTH 1-24): 24 amino acid peptide Deslorelin: 10 amino acid peptide Desmopressin: 9 amino acid peptide Elamipretide (SS-31): 4 amino acid peptide Epitalon: 4 amino acid peptide GHK Cu (Copper peptide): 3 amino acid peptide GHRP-2: 5 amino acid peptide GHRP-6: 6 amino acid peptide Glutathione: 3 amino acid peptide Insulin*: 51 amino acid peptide |
Ipamorelin: 5 amino acid peptide Kisspeptin 10: 10 amino acid peptide Leuprolide: 9 amino acid peptide Liraglutide: 32 amino acid peptide Melanotan II: 7 amino acid peptide MOTS-c: 16 amino acid peptide Oxytocin: 9 amino acid peptide Semaglutide: 29 amino acid peptide Sermorelin: 29 amino acid peptide Sincalide: 8 amino acid peptide Tesamorelin Acetate*: 44 amino acid peptide Tirzepatide: 39 amino acid peptide Thymosin Alpha 1: 28 amino acid peptide Thymosin Beta 4*: 43 amino acid peptide
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* At the time of this writing, the Food and Drug Administration (FDA) will not allow compounders to prepare formulations classified as
“biologics”, which are defined to be those exceeding 40 amino acids.
Beyond these peptides there are many others which, over time, will be found useful in treating a wide array of medical issues. Whatever the next ones will be, CIAL is confident in our ability to develop methods for testing their potency, beyond use dating, sterility, endotoxin levels, and more.
As we turn our attention from a general understanding of peptides to more practical considerations related to compounding, we have found the following information to be helpful guidance to our customers…
Solubility
Peptides which are formulated as aqueous injections can demonstrate solubility difficulties, especially those which are more hydrophobic. This is because they contain many aromatic amino acids such as phenylalanine, tyrosine, or tryptophan, or those having non-ionic side chain amino acids such as leucine, isoleucine, or lysine. These must be given more time for dissolution and checked carefully for any undissolved powder before undergoing sterile filtration. Those which are more hydrophilic will readily dissolve in water or saline. Generally, mixing in a beaker on a magnetic stirrer should work fine but it is best to avoid vortexing, vigorous shaking, or placing in a sonic bath. These actions can break down those larger peptides (10+ amino acids).
Lyophilization and Freeze/Thaw Cycles
As with many higher molecular weight active pharmaceutical ingredients (API’s), there is the possibility that during freezing steps, ice crystal formation of aqueous solutions will cause mechanical breaking of peptide molecules. It can be expected that a loss of potency (~5%) of the peptide to occur for each freeze/thaw cycle. Many of our compounders have found it useful to add extra API to compensate for this loss. Multiple freeze/thaw cycles are not recommended.
If the compounded product will be delivered as a lyophilized cake in an injection vial, it is recommended that some water remain in the cake (1 – 2%). It should not be thoroughly dried in a hard vacuum, but rather the freeze-drying vacuum process be slower and gentler as often used for small molecules.
Storage
Whether dry, lyophilized, or as an injection, most peptides do best when the final dosage form is stored under refrigeration (2º - 8ºC).
pH
As a general rule, peptides tend to be more stable in neutral to slightly acidic solutions. Although individual examples may vary slightly, some examples CIAL has found include; Semaglutide appears to be more stable at pH 7.4, Tirzepatide can precipitate under acidic conditions, and Liraglutide and Semaglutide can break down in lower pH solutions. In an aqueous solution, buffering the pH is recommended. Solid, non-aqueous forms do not typically break down as easily, thus, should be less pH sensitive. With very few exceptions, nearly all peptides are sensitive to heat and oxidation.
Peptides and other API’s
Surprising things can happen when adding other actives to a peptide preparation. Some examples we have found; the addition of Methylcobalamin, Hydroxocobalamin, Pyridoxine HCl, or Pyridoxal 5 Phosphate to Semaglutide can negatively affect the stability of aqueous formulations. The combination of these API’s appear to bind or react with Semaglutide in a way that reduces its potency. Without laboratory test data, these interactions would not have been known.
Calculations
As many peptides to no have USP monographs, reviewing the peptide CoA can be confusing. Assay values and other information affecting potency can be highly varied because the information is inconsistent between manufacturers. For instance, some report Purity % or Related Substances % while others do not. In short, the goal in determining the Potency value should be based upon the amount of the bare peptide and should remove values from, but not limited to; water, solvents, salts, and acids.
Sterile Filtration Issues (Plastic vs Glass)
It is common for peptides to absorb onto the surfaces of microporous membrane filters. The type of filter membrane can also make a difference. While CIAL prefers to use polypropylene or wettable PTFE membranes in polypropylene housings, others can also work but may absorb more of the peptide. A good technique during sterile filtration step is to waste the first 1-2 mL, then begin collecting the solution in glass vials. This technique works well because during the wasting step, you are allowing the filter to bind whatever amount of peptide that it will. After this has been accomplished, it should not retain more, so the remaining solution should be fully potent.
In addition, most all peptides will cling to plastic surfaces, to some extent. While the amount is generally minimal, we have seen some that will retain significantly more (>5%). Use of glass beakers and vials has not yet been found to be a problem, but use of plastic (ie: syringes), can often reduce the peptide concentration in an aqueous solution. Adding more peptide to the preparation to compensate, can be a helpful remedy.
While on this topic, CIAL utilizes biocompatible UHPLC instruments which are specially modified for potency testing of peptides. This is done by eliminating use of all stainless steel in the instrument tubing which can potentially bind up many proteins and peptides. If not accounted for, a peptide’s value could be skewed resulting in a subpotent recovery when in fact it is not. As a result, CIAL is very cautious in each analytical step taken to ensure the CoA’s we release are as accurate as possible.
Conclusion
CIAL’s primary focus is to assist compounders in making successful and accurate (peptide) products that will safely, and effectively, provide the desired result(s) to their patients. We understand that our success stems from your success. Due to the highly variable “personalities” among peptides, we strongly suggest compounders have their initial preparations tested to be sure they are made as intended. Having successfully tested a wide array of peptides in various formulation types, CIAL hopes the information provided will prove helpful.
What is Water Activity?
To have BUDs longer than these dates is possible (up to 180 days), but there must be stability indicating test data to support this. Our laboratory routinely performs such stability testing. Please contact us if you are interested in having your formulations tested to be able to extend your BUDs.
So, what is water activity and why is it important?
What is water activity? Is it the amount of water in the product, similar to the number we often see on the certificate of analysis for an active ingredient? Is it the same as loss on drying (LOD)? The answer is “No” to both of these questions.
Water activity (Aw) is the ratio of the vapor pressure of water in a preparation vs. the vapor pressure of pure water at the same temperature, in a closed system. Or for a simpler way of thinking about it, we can refer to it as being related to a measurement of the humidity within a container of product. Water activity can be measured using specialized equipment and the number generated will relate to humidity within the preparation. Typically, water activity values will range from 1.0 on the high end, to near zero on the low end. These numbers would be examples of a totally aqueous solution, and a dry powder, respectively.
The higher the Aw value, the greater the opportunity for microbial growth and the greater the opportunity for some active ingredients to breakdown due to hydrolysis. So, if water activity can be reduced, the BUD may be able to be extended. In addition, with lower Aw values, antimicrobial effectiveness of preservatives is improved. Susceptibility to microbial contamination of multiuse products which might be contaminated by the user, such as topical creams, is reduced with lower Aw values. Also, the life of many active ingredients is extended. Beyond these, the frequency to perform Microbial Enumeration Tests <61> and Tests for Specified Microorganisms <62> can be reduced.
To understand the importance of water activity values, it is important to understand that many bacteria will not survive in products having water activity below 0.86. Most molds and yeast will not grow if water activity is below 0.77 although some can grow with Aw down to 0.60. Therefore, a good target for water activity which will optimize shelf life insofar as microbial growth, is to achieve Aw values of 0.60 or lower. This number means the vapor pressure is 60% of that of pure water.
How can water activity be reduced?
Certainly non-aqueous preparations or dry solid dosage forms will not support microbial growth or spore germination because of their inherent low water activity. For dosage forms containing water, one way to reduce water activity, of course, is to reduce the amount of water in the formulation. This might not always be practical however. Consider an aqueous oral solution or suspension which must contain a considerable amount of water in its formulation. Consider also, some topical products which need a certain quantity of water to formulate properly or to provide needed characteristics. So we might ask, in what way can these be reformulated to reduce the Aw value? It turns out that small changes in the concentration of alcohol, glycerin, or propylene glycol can reduce water activity by linking to water molecules by means of hydrogen bonding. Likewise, the addition of sucrose or sodium chloride can reduce water activity and make preparations self-preserving. Possible, also, is the addition of solids in a formulation, especially those which absorb or bind water, such as methylcellulose, will reduce the Aw value. In your formulations, consider if these or similar substances would be appropriate and if so, it just might make a significant difference in its BUD.
Final comments.
If you would like to determine the Aw value of any of your preparations so you will know what BUD you can use, please send us a sample for testing. We have the specialized instrumentation to perform this test for any dosage form. If you have a desire to modify a formulation in an effort to reduce its Aw value and thus extend its BUD, this test result will establish its baseline value before any formulation changes are made. Then consider the suggestions for reducing the Aw value provided above, reformulate, and have us retest to identify the effectiveness of your modification(s).
Methylcobalamin and Light
Vitamin B12 is a common nutritional supplement in many formulations. The three most often used
forms are Cyanocobalamin (B12), Methylcobalamin (Me-B12) and Hydroxocobalamin (B12a).
When formulating methylcobalamin, many are aware of its sensitivity to light but may not know just how
sensitive it is. In our lab we carried out an experiment to see just how sensitive a typical formulation
might be to typical interior lab lighting. The formulation we selected as our test sample was one
containing methylcobalamin 1000mcg/ml in normal saline plus benzyl alcohol 2%. We assayed the
sample to check its potency and found it to be 985mcg/mL so it was 98.5% potent, thus meeting
requirements. We then exposed the solution in a clear glass vial for 5 minutes under our lab’s typical
florescent lighting, then once again tested it for potency. The potency was now at 54.4%, a 44.1%
decline! Much of the active had converted to hydroxocobalamin (B12a). After another 5 minutes
exposure, now a total of 10 minutes, the potency was now down to 30.8% a 67.7% decline from the
initial potency. After a total of 15 minutes, the potency stood at 15.2%, a total decline of 83.3% from the
starting point! Clearly, methylcobalamin is VERY light sensitive and easily converts to
hydroxocobalamin.
To maintain potency all precautions possible to protect it from light should be taken. In many labs,
amber glassware is used and this works quite well. Another possibility is to wrap all glassware with
aluminum foil. This works but is awkward because the sample cannot be inspected to make sure all of
the active has dissolved nor can volumetric graduations be seen to verify proper dilution has been
accomplished. (Please note: when weighing out the dry API powder, methylcobalamin is fairly light
stable so this step can be completed in normal room light. Once it has been put into solution however,
it becomes very light sensitive.)
Compounding Suggestions:
We found we could prepare and test a methylcobalamin formulation successfully by turning off the lab
lights and operate by the small amount of light coming into the lab from a distant window. The work
area was quite dark, glassware graduations could just barely be read, but the technique does work,
methylcobalamin did not break down to any measurable degree. Unfortunately, others in the lab didn’t
appreciate the idea so much!
More recently, we found we could use a red light bulb in a separate area of our lab and the potency did
not decline at all, even after 15 minutes exposure time. The bulb we used was a Philips LED 8 Watt
bulb which was purchased at our local Home Depot for a very nominal cost. We repeated our
aforementioned 5, 10 and 15 minute test with this bulb as the only light source and found it worked very
well, no breakdown occured. Our analyst had plenty of light to carry out all our normal testing
procedures, could easily read glassware graduations, and best of all, didn’t upset the others!
Water, Water, Everywhere!
Here at Compounder’s International Analytical Laboratory we find that the vast majority of the
formulations we test are good, having potencies which fall within the required range.
Occasionally, however, a potency comes in low and we always test the sample again to see if
the number(s) change or if they repeat. If the low potency value repeats there could be several
reasons for this and among them is the presence of water or a residual solvent in the active
ingredient.
On the Certificate of Analysis which is supplied with the active ingredient, there is a line which
provides the % Potency of the compound. Nearly always, this potency is given on the
anhydrous basis. This means the powder was dried before it was tested, or the amount of
water or residual solvent was determined and its value was subtracted from the weight of the
powder before it was assayed and the potency determined. Per the USP, there is a maximum
allowed % Water or % Loss on Drying (LOD) for each active ingredient. Usually, this number is
less than 1% so might be ignored with little consequence. But for some actives the amount is
significant and can seriously lower the potency of the final formulation since some of the powder
weight is just water. A few examples of this include:
Azithromycin will have between 4.0 to 5.0% water
Bacitracin can have up to 5.0% LOD
Betamethasone Acetate can contain up to 4% water
Bupivacaine HCl will have between 4.0 and 6.0% water and up to 2% solvent
Ciprofloxacin will have 4.7 and 6.7% water
Clindamycin HCl contains 3.0 to 6.0% Water
Dexamethasone Sodium Phosphate can have up to 16.0% total water and alcohol
Folic Acid will contain up to 8.5% water
Levothyroxine Sodium (T4) can contain as much 11.0% water.
Liothyronine Sodium (T3) can have as much as 4.0% water
There are many more examples, but you get the idea.
When compounding, it is always a good practice to note the % Water or % LOD and to subtract
this value from the anhydrous % Potency to determine the actual “as is” potency of the active.
In the case of the T4 example above, if the anhydrous potency is 98.3% and the % Water is
9.8%, the actual potency of this powder is 88.5%, so this new value should be used to develop
the formulation. Therefore, if the required amount of T4 to make a 1:100 dilution is 1.00gram,
then divide the 1.00gram by 0.885 (88.5%) to find that the weight required to achieve the correct
concentration, which would be 1.13grams in this case.
Compounding Suggestions:
We like to suggest that whenever a new batch of active is received, the real “as is” potency
should be calculated from the anhydrous % Potency and the % Water or % LOD and written on
the front of the container before it is placed in stock. (In fact, even when there is not a
significant % water, there might be a lower potency value anyway, such as 95% or 97%). This
written potency value would then serve as a reminder as to the actual potency of each
ingredient when being used, and the weight adjusted to obtain a more accurate formulation.
Why are T3/T4 Formulations Difficult to Make?
Liothyronine Sodium (T3) and Levothyroxine Sodium (T4) are iodonated amino acids and are typically
formulated in microgram concentrations. These active pharmaceutical ingredients (APIs) usually
contain a significant amount of water: up to 4% in the T3 powder and up to 11% in the T4 powder. The
primary reasons for the difficulty in preparing formulations containing T3 and/or T4 arise from these 3
issues; microgram concentrations, iodination of the compound, and water.
So, what makes these 3 issues so problematic?
1. In the preparation of, lets say, a powder blend to be use for preparation of T3/T4 capsules, typically
the active(s) are mixed with microcrystalline cellulose and perhaps methyl cellulose as well, to provide
the correct dilution and desired slow release properties. Sometimes a coloring agent is also included to
provide a visual indication of completeness of mixing. Normally, these are blended according to the
technique of geometric additions and mixed/stirred in the same fashion as many of the other capsule
formulations. But, there’s just one problem. In this case, we are not blending milligram amounts of an
active, we are blending microgram amounts. If we can visualize how much 1 milligram of powder is,
then imagine how much 1/1000th of this would be. We can begin to realize it would only take a few
specks of the API powder to equal a microgram. These few specks must be evenly distributed
throughout the powder blend. It may seem counterintuitive, but it actually takes longer to evenly blend
micrograms of an ingredient than it does for milligrams. In addition, if the particle size of the API is
significantly different from that of the other ingredients, once they are well mixed they might not stay
that way. During handling, the vibrations generated in the process will cause the smaller particles to
move toward the bottom of the container while the larger one will migrate to the top. (Consider what we
observe when we open a new box of granola cereal. The big clusters are usually at the top while the
crumbs are at the bottom).
2. Those that took general and/or organic chemistry class in school may recall the halogens, listed
near the right hand column of the periodic table, tend to be strongly electronegative because of their
electron rich outer shell orbital. T3 and T4 both contain iodine atoms, members of this halogen group.
If the powder blend described above is accomplished in a plastic container, the agitation of the cellulose
along with the T3/T4 actives will develop a strong static charge on the walls of the container which will
attract these microgram amounts of ingredients out of the powder and onto the surface of the container.
The more rapidly the powder blend is mixed, the stronger the static charge. In our practice we have
observed a prominent light brown color of the T4 powder clinging to the container and when we have
tested the wall scrapings, they were 30-40% higher in both the T3 and the T4 than within the main
volume of powder.
3. Since a fairly significant portion of the weight of the T3 and T4 powder is water (up to 4% and 11%
respectively), adjustments in the desired amounts of these ingredients must be made to account for
this. (See our article entitled “Water, Water Everywhere”). Unless an appropriate extra amount of the
API powder is included in the formulation, the final product will be sub potent.
Compounding Suggestions:
Formulations containing microgram amounts of T3 and or T4 actives must be blended longer and more
completely than usual so as to account for these very low concentrations. The blending should be
accomplished in either glass or metal containers, not plastic, and should have a tumbling action of side
over side and end over end to make sure the actives are well mixed. Mortar and pestle mixing seldom
will be sufficient. The time required will vary depending upon batch size and mixing speed, but a 2 hour
mixing time would not be unusual. Ideally, both the active and inert (excipient) ingredients are
micronized to approximately the same particle size to prevent size stratification within the batch once
the blend is finished. And finally, care should be taken to account for the % water content in the API
powders.
HCG Formulation Facts
Human Chorionic Gonadotropin (HCG) is a protein containing a total of 237 amino acids and made up
of 2 subunits, an alpha chain and a beta chain, which are held together by the weak forces of hydrogen
bonding. Apparently, the protein activity requires the intact molecule which is lost or greatly reduced if
these subunits break apart. In addition, because of its amino acid sequence and 3-dimensional
orientation, the outer surface is highly charged ionically.
The official assay method which is spelled out in the USP, involves dosing laboratory rats, then
sacrificing them and determining the weight of their uteruses, which can be correlated to the potency of
HCG. Since this is a tedious, time consuming and distasteful process, it is not practical on a day to day
basis. One of the alternate methods which has been used to assay the concentration of HCG, is by
means of an immunoassay technique. This utilizes monoclonal antibodies that are specific to a specific
sequence of amino acids found in the beta subunit of the protein. Such a technique works best for invivo
testing because, in the body, the protein tends to maintain its structural integrity. Under in-vitro
conditions however, such as a pharmaceutical preparation, this method can produce false high values
because it will label both the intact protein as well as the disassociated beta subunit, and certain
fragments of this subunit, as if they are the active intact form. Like all proteins, HCG is fragile and can
be broken apart and denatured by processing, mechanical mixing, shaking, homogenizing, or ultrasonic
dissolution. This can occur during the supplier’s manufacturing processes as well as during
compounding processes.
In our lab, we have developed and validated a chromatographic method which separates the intact
protein from its subunits, as well as from various peptide pieces. This allows us to correctly assay only
the intact protein concentration, which correlates well with its potency.
Compounding Suggestions:
HCG has a tendency to adhere to plastic more than glass, primarily when in solution. This becomes
especially significant in concentrations below 1,000 units/mL. To help with this, we suggest not using
plastic materials for anything that might come into contact with the HCG solution. (As a dry powder,
this is not as critical). Also, for all glassware that will be exposed to solutions of HCG, pre-rinsing then
wasting, with some of the HCG solution will help prevent losses. Like most proteins, some of it can
adhere to filter materials. When sterile filtering, therefore, we suggest using a low-protein binding filter
and wasting the first 1-2 mL that passes through the filter, then begin dispensing into sterile containers
after that. HCG is sensitive to warm temperatures, so both in the solid form as well as in solution, it
should be stored and shipped under refrigeration. Freezing, especially freezing then thawing and
refreezing of any protein solution is not suggested because ice crystals, as they form, can damage the
molecule. In our experience, we find that when HCG is dry mixed (often with mannitol powder) it is best
accomplished by tumbling in a plastic or glass container. Like most proteins its exposure to metal
should be limited. It is best not to mix in a mortar and pestle, which can cause fragmentation of the
protein due to the inherent mechanical grinding action of this technique. And, finally, when making and
mixing solutions of HCG, do not shake or sonicate. Instead, swirl or rock the container in a smooth
action to avoid breaking the protein.