Why Reconstituting IGF-1 Peptides w/ Acetic Acid has its Advantages

Often, you read commenters on bodybuilding & biohacking platforms suggesting that simply reconstituting your lyophilized IGF-1 analogs with bacteriostatic water will suffice, and while this may confer peptide stability for some period of time, the extent of that period of time is unbeknownst to any of us due to a total lack of literature on the matter. If a researcher wants a solution that is intended to stabilize the contained peptides beyond just a slew of days, opting for 0.6% acetic acid solution might be more advantageous. This is because the acidity of the solution containing the peptide is critical in determining how stable the peptide will remain. According to Merck, there are at least five potential degradation pathways for peptides, and of these five, two pathways involve base-catalyzed reactions, while another pathway becomes more & more likely as the containing solution grows more basic1. With that written, it is necessary to note that bacteriostatic water does possess some qualities that do offer some protection to the peptides it will mix with in that respect: it has a pH range of 4.5-7.0, with an average at 5.7, meaning it is slightly acidic (neutral pH is 7, with pH <7 being acidic, and pH >7 being basic). However, a 0.6% AA solution boasts a pH of approximately 2.87;

0.6% Acetic Acid pH Calculation

So comparatively, a 0.6% AA solution is 682-fold more acidic than bacteriostatic water, which could theoretically lead to enhanced stability of the dissolved peptide, granting the researcher more time to consume any given prepared vial. This expanded longevity is demonstrated by an IGF-1 product formulated by Cerilliant that contains a comparable solution acidity (pH = 2.6 in this case, owing to a higher AA concentration of 2%)7. Notably, this solution has been demonstrated on analysis to preserve ~97.8% of the native (unmodified) form of IGF-1, and its producers indicate that it is capable of being refrigerated for 8 weeks;

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Most researchers will not opt to spend eight weeks dosing out of a reconstituted vial of IGF-1, as most experiments typically last 4 weeks & under. But perhaps confidence can be derived from knowing that a solution only 1.58-fold more acidic than a homemade iteration consisting of 0.6% AA can survive that long in a refrigerator.

Acetic Acid as an Antiseptic Agent

In a experiment carried out by Fraise et. al. involving Gram-positive & Gram-negative bacteria, acetic acid was found to inhibit bacterial growth at concentrations as low as 0.166%, several fold lower than the 0.6% typically utilized to reconstitute lyophilized IGF-1 variants.

Fraise et. al.
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In a study carried out by Trzaska et. al. testing AA’s fungistatic & fungicidal capacities across four species of the Mucorales fungal order (known to induce mucormycosis, which is fatal in >90% of established infections), concentrations of AA as low as 0.3% were found to exhibit potent antifungal effects. The authors were also able to reveal that these antifungal properties are not solely due to the pH of the solution, as other organic & inorganic acids of similar pH did not exhibit as profound an effect.

Trzaska et. al.
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In an in vitro study carried out by Alphin et. al., AA at concentrations of 1 & 3% were found to inactivate influenza on metal surfaces. Separately, in an in vivo study carried out by Pianta et. al., a group of COVID-19-infected individuals treated with 0.34% AA inhalation experienced improvement in symptoms relative to the group that did not receive the inhalation treatment.

Alphin et. al.
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Pianta et. al.
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Now, this is not to discredit bacteriostatic water (0.9% benzyl alcohol) as an antiseptic agent in its own right. But every antiseptic agent differs in its effectiveness against the colossal spectrum of pathogens that exist. While a pathogen may exhibit vulnerability to one antiseptic agent, it may, at the same time, demonstrate a degree of resistance to another antiseptic agent. Based on this reality, it may be advantageous to utilize both AA & bacteriostatic water, instead of simply relying on one or the other. Indeed, this is how Increlex, an FDA-approved recombinant IGF-1 peptide, is formulated, albeit utilizing the conjugate base of acetic acid (which should theoretically still display some degree of antiseptic traits, as described by Trzaska et. al.);

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Ultimately, reconstitution with 0.6% AA is an exceedingly simple task, even if it adds some steps to the process. This video demonstrates that. Indulge.

Going Above & Beyond; Opting for a Lower-Concentration Reconstitution Solution

While 0.6% AA is about as tried & true as a reconstitution agent can be, there may be room to formulate a lower concentration that could maintain similar antiseptic capacities, while additionally conferring benefits on two fronts: peptide stability and injection pain. We know from references detailed above that 0.3-0.312% AA has been proven to be effective as an antiseptic against a wide range pathogens, including bacteria & fungi. However, the highly resistant MRSA requires a leap up to a minimum concentration of 0.625% for growth inhibition, which is greater than the 0.6% traditionally employed for IGF-1 peptide reconstitution. Based on this detail, any concentration of AA greater than or equal to 0.312% would theoretically inhibit many of the same pathogens that a 0.6% solution otherwise would. Understanding that a lower concentration of AA can maintain antiseptic functionality, we can now explore potential benefits with regards peptide stability & injection pain, and we’ll do this by utilizing a solution of 0.333% AA as an example.

Peptide Stability

Earlier in this article, a lower pH was described as generally being conducive to peptide stability, and this is arguably proven by Cerilliant’s IGF-1 formulation, with a pH of 2.6, that demonstrated 97.8% purity on its Certificate of Analysis, and that allegedly lasts for 8 weeks in a refrigerator. With this written, however, one possible peptide degradation pathway should be acknowledged: deamidation. Deamidation occurs with Asn or Gln amino acid residues, of which, the IGF-1 peptide has 1 & 2 of, respectively. Now, to be clear, deamidation occurs much more frequently in neutral or basic conditions than acidic, as Bhatt et. al. demonstrated in an experiment that compared a range of pH’s & buffer concentrations to determine differences in the rates of deamidation of the singular Asn residue of the adrenocorticotropic hormone (ACTH);

Bhatt et. al.
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Acidic pH is clearly favorable for peptide stability in the face of deamidation pressure when compared to neutral or basic solutions. But what happens to the observed rate of deamidation when different acidic pH’s are compared amongst one another? Joshi et. al. tested this by measuring the rate of deamidation occurring to the Asn & Gln amino acid residues of a glucagon hormone peptide fragment (22-29) across a pH range of 1-3, finding that there appears to be a counterintuitive gradient wherein the more acidic the solution, the faster the rate of deamidation;

Joshi et. al.
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Reconciling both Bhatt et. al. & Joshi et. al.’s findings, it may be safe to assume that, while an acidic pH is preferable as far as peptide stability goes, a pH floor of >3 may present the most promise in keeping a reconstituted peptide in its native, unmodified form. Conveniently, a 0.333% AA solution boasts a pH of 3.

Pain Associated with Injection

In a scoping review on buffers as they pertain to protein formulation, Zbacnik et. al. deduce that both buffer concentration & pH can determine whether an injection will be more likely to produce pain or not, which in turn could jeopardize patient adherence to a dosing protocol. The study authors note that the lower the buffer concentration, and the closer the buffer’s pH to the body’s physiological pH (7.4), the lower the likelihood of injection-associated pain. This assertion on pH, in particular, derives in part from a study carried out by Klement et. al. that found that intravenous injection pain was associated w/ pH levels below 4 & above 11.

Zbacnik et. al.
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Klement et. al.
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Opting for a reconstitution agent consisting of 0.333% AA as opposed 0.6% would potentially reduce injection-associated pain along both veins: by reducing the buffer concentration by nearly half, and by reducing the acidity by approximately 25% (pH 2.87 -> 3). Of course, bacteriostatic water should be used to draw the IGF-1-peptide-containing AA solution, thereby raising the pH of the injected bolus in its own right, but starting from a pH of 3 as opposed to 2.87 will certainly make the final injection aliquot less acidic.

References

  1. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/research-and-disease-areas/cell-and-developmental-biology-research/peptide-stability
  2. Fraise AP, Wilkinson MA, Bradley CR, Oppenheim B, Moiemen N. The antibacterial activity and stability of acetic acid. J Hosp Infect. 2013 Aug;84(4):329-31. doi: 10.1016/j.jhin.2013.05.001. Epub 2013 Jun 7. PMID: 23747099.
  3. Trzaska WJ, Correia JN, Villegas MT, May RC, Voelz K. pH manipulation as a novel strategy for treating mucormycosis. Antimicrob Agents Chemother. 2015 Nov;59(11):6968-74. doi: 10.1128/AAC.01366-15. Epub 2015 Aug 31. PMID: 26324263; PMCID: PMC4604374.
  4. Alphin RL, Johnson KJ, Ladman BS, Benson ER. Inactivation of avian influenza virus using four common chemicals and one detergent. Poult Sci. 2009 Jun;88(6):1181-5. doi: 10.3382/ps.2008-00527. PMID: 19439628.
  5. Pianta L, Vinciguerra A, Bertazzoni G, Morello R, Mangiatordi F, Lund VJ, Trimarchi M. Acetic acid disinfection as a potential adjunctive therapy for non-severe COVID-19. Eur Arch Otorhinolaryngol. 2020 Oct;277(10):2921-2924. doi: 10.1007/s00405-020-06067-8. Epub 2020 May 24. PMID: 32449022; PMCID: PMC7245632.
  6. https://www.ipsen.com/products/Increlex_Full_Prescribing_Information
  7. https://www.cerilliant.com/shopOnline/COA.aspx?itemno=ba0015a9-ce61-4ec3-be57-c298f3db702c&lotno=FN11062013
  8. Zbacnik TJ, Holcomb RE, Katayama DS, Murphy BM, Payne RW, Coccaro RC, Evans GJ, Matsuura JE, Henry CS, Manning MC. Role of Buffers in Protein Formulations. J Pharm Sci. 2017 Mar;106(3):713-733. doi: 10.1016/j.xphs.2016.11.014. Epub 2016 Nov 26. PMID: 27894967.
  9. Klement W, Arndt JO. Pain on i.v. injection of some anaesthetic agents is evoked by the unphysiological osmolality or pH of their formulations. Br J Anaesth. 1991 Feb;66(2):189-95. doi: 10.1093/bja/66.2.189. PMID: 1817619.
  10. Bhatt NP, Patel K, Borchardt RT. Chemical pathways of peptide degradation. I. Deamidation of adrenocorticotropic hormone. Pharm Res. 1990 Jun;7(6):593-9. doi: 10.1023/a:1015862026539. PMID: 2164192.
  11. Joshi AB, Kirsch LE. The relative rates of glutamine and asparagine deamidation in glucagon fragment 22-29 under acidic conditions. J Pharm Sci. 2002 Nov;91(11):2331-45. doi: 10.1002/jps.10213. PMID: 12379918.