measured pH of various peptides

[Sera]

Thought this might be worth posting as I’ve done some measurements of some various peptides I have in the fridge.

SSA tirz 15 (feb 19): pH 5.9 (unbuffered?)
SSA reta 10 (jun 30): pH 7.3
SSA hgh 24iu (no date but relatively recent): pH 6.6
Lobster hgh 31iu (no date but relatively recent): pH 7.1

Goes to show that there are actually buffers in most of these other than the earlier manufactured tirzepatide.

 

[Ateam2023]

Thought this might be worth posting as I’ve done some measurements of some various peptides I have in the fridge.

SSA tirz 15 (feb 19): pH 5.9 (unbuffered?)
SSA reta 10 (jun 30): pH 7.3
SSA hgh 24iu (no date but relatively recent): pH 6.6
Lobster hgh 31iu (no date but relatively recent): pH 7.1

Goes to show that there are actually buffers in most of these other than the earlier manufactured tirzepatide.

i'm being lazy, but whats the acceptable range of ph in a particular peptide?

 

[Sera]

i'm being lazy, but whats the acceptable range of ph in a particular peptide?

Well for hgh it seems about 6.5-7ish is optimal.
Other peptides like glp-1s I don’t have specific information about but probably around 7 is a safe bet.
Some peptides prefer acidic conditions like cagrilintide though.

I should mention that all these were done with bac water.

 

[psauce]

This isn't an especially meaningful measure. There's a basic issue here, which is that peptides don't have pH. The aqueous solution they're dissolved in does, though, and that's what you measured.

What's more, amino acids have both acid and basic side chains. Aspartic acid is the most acidic (pKa ~ 3.1), while arginine is the most basic (pKa~12.5). These are approximate values because the tertiary structure of the protein can hide or expose residues, which makes them more or less available for (de)protonation.

This means proteins in solution tend to act as their own pH buffers. The side chains that are acidic will deprotonate, the ones that are basic will suck up the excess protons. Aqueous solutions of dissolved protein tend to have a pH of around 6-7 almost always for this reason.

Extreme pH values can cause acid or base hydrolysis, but we're talking pH > 13 or pH < 1, and in a lab setting hydrolysis is performed in closed tubes incubated at 110 C even with these pH extremes.

What's more, once the injection hits body fluid, carbonic anhydrase takes over and nothing else matters. You could inject a peptide in a fluid acidic enough to do a number on your tissue and the peptide would still work just fine.

 

[Sera]

This isn't an especially meaningful measure. There's a basic issue here, which is that peptides don't have pH. The aqueous solution they're dissolved in does, though, and that's what you measured.

What's more, amino acids have both acid and basic side chains. Aspartic acid is the most acidic (pKa ~ 3.1), while arginine is the most basic (pKa~12.5). These are approximate values because the tertiary structure of the protein can hide or expose residues, which makes them more or less available for (de)protonation.

This means proteins in solution tend to act as their own pH buffers. The side chains that are acidic will deprotonate, the ones that are basic will suck up the excess protons. Aqueous solutions of dissolved protein tend to have a pH of around 6-7 almost always for this reason.

Extreme pH values can cause acid or base hydrolysis, but we're talking pH > 13 or pH < 1, and in a lab setting hydrolysis is performed in closed tubes incubated at 110 C even with these pH extremes.

What's more, once the injection hits body fluid, carbonic anhydrase takes over and nothing else matters. You could inject a peptide in a fluid acidic enough to do a number on your tissue and the peptide would still work just fine.

Appreciate the thoughtful comment.

Of course I’m measuring the pH of various peptide formulations, not the peptides themselves.

A key point is that a peptide’s isoelectric point (pI) - where it would “buffer itself” to in unbuffered solution - is often where it’s least soluble and most hydrophobic. That’s because net charge hits zero there, minimising repulsion and letting hydrophobics drive aggregation, precipitation, or adsorption to filtration membranes.

For example, benzyl alcohol in bacteriostatic water at 0.9% w/v is acidic and overpowers any small buffering capacity the peptide has, dragging everything to ~pH 5.7 if unbuffered. For some peptides, that might be fine, but each has a different ideal pH - you don’t want to just let BAC water set it and leave it.

GLP-1 agonists are a great example: They have fatty tails that can cause solubility issues if pH is wrong. To maximise dispersion, we want to reduce protonation of those tails (e.g., by shifting pH above ~7, where they stay charged and repel each other). Protonated tails become more hydrophobic and prone to clustering, and these aggregates could possibly be immunogenic. Note that the peptide’s pI isn’t necessarily the same pH that optimises tail dispersion.

HGH is another key example where pH buffering matters - you’d have a bad time at 5.7 (near its pI of 5.27), with high aggregation risk.

So I hope you see now why pH control is crucial for pharmaceutical peptide and protein formulations!

 

[psauce]

I realized as I was falling asleep last night that I had failed to consider the outsized effect of isoelectric point in polypeptides versus larger proteins. Very elementary error. I appreciate the thorough response.

 

[Sera]

I realized as I was falling asleep last night that I had failed to consider the outsized effect of isoelectric point in polypeptides versus larger proteins. Very elementary error. I appreciate the thorough response.

Honestly I haven’t been into this topic for too long, so your post prompted me to do some more research to try to support myself and therefore learn some more in the process.