The FDA makes the case for filtering peptides

[Ghoul]

Thx for reviewing those key pts.

Small point: Technically, since (I assume) Jano is basing purity relative to the initial vial sample mass, he doesn't really "miss" any impurities (purity = HGH found / sample mass). We just can't say much about the nature of all those impurities based on the dimer content, although lower dimer would hopefully have some relation to immunogenicity, and good lab practice... So his results do reflect what you're injecting - he just isn't telling us much about what's in there other than the HGH and "dimers" making it thru 0.2 µm filter.

No, it's not compared to the initial vial sample mass.

The vial is reconstituted with X amount of water, a sample is withdrawn, filtered, injected into the HPLC column, separated into the various components, and the total area under the peaks of all components is compared to the area under the peak that identifies the target peptide to determine purity.

Only the post-filtration peptide sample is analyzed.

Researchers have the advantage of knowing exactly how much peptide was put into a vial, and can extrapolate how much was lost in the filter based on the amount of "missing" mass, and sometimes use that to estimate how much aggregation has taken place.

 

[Ghoul]

Thx for reviewing those key pts.

Small point: Technically, since (I assume) Jano is basing purity relative to the initial vial sample mass, he doesn't really "miss" any impurities (purity = HGH found / sample mass). We just can't say much about the nature of all those impurities based on the dimer content, although lower dimer would hopefully have some relation to immunogenicity, and good lab practice... So his results do reflect what you're injecting - he just isn't telling us much about what's in there other than the HGH and "dimers" making it thru 0.2 µm filter.

Let me elaborate on this, and what brought this topic up. In a study of rHGH instability (which takes the form of aggregation), after abusing the shit out of the rHGH the "purity" results from HPLC (SEC, Size exclusion chromatography) were coming back as 98% and 96%, or basically, no degradation.

After accounting for the "missing" rHGH, which must have become aggregates unable to pass through the filter/column, the corrected purity measurement was 68% and 81% .

But this can only be done if you have a starting point of knowing precisely how much monomer was put into the vial to start with. Something that's an unknown with UGL and varies from vial to vial.

PS: Also, I didn't want to over complicate things, but even aggregates that are smaller than the .22um syringe filter don't get measured if they're insoluble (as most are), because they get stuck in the chromatography column.

Dimer, (two peptide monomers), oligomer, and slightly larger aggregates are soluble, but most beyond that are not.

 

[Sera]

I use filters with the Supor PES membrane (Pall, Braun, and others) which specify they're shed-free, and for use in pharma compounding, IV, bolus, and even interocular (eye) injection filtration.

Since shedding particles would also foul samples for chromatography analysis (likely the most common use for these filters) one would assume the large brands that supply labs don't have this issue, like Pall, Tisch, Cobetter.

The no name Amazon and Alibaba filters might not be as good quality though.

Nothing wrong with pre-wetting filters though. Although peptide loss to adhesion is usually very low with PES, pre wetting probably lowers it more and avoids increasing the peptide concentration since some water remains behind while the peptide passes through when absorbed by the dry filter.

View attachment 323547

Do you have any specific recommendations, as the prices are eye watering for some of these.

 

[RandGuy]

No, it's not compared to the initial vial sample mass.

The vial is reconstituted with X amount of water, a sample is withdrawn, filtered, injected into the HPLC column, separated into the various components, and the total area under the peaks of all components is compared to the area under the peak that identifies the target peptide to determine purity.

Sorry for my ignorance (new to this stuff)! Was misreading Jano's numbers:

where then the mg reflect the total measured rhGH without regard to what else might be in the vial, and the % purity only applies to what makes it through the column after filtering (seems like a pretty sketchy way to measure "purity" of sample). So, just as you said, the notion that "97%" even roughly represents something about the purity of what you're injecting assumes that you've done 0.2 µm filtering.

 

[RandGuy]

Do you have any specific recommendations, as the prices are eye watering for some of these.

I ordered one of these filter kits just to get started (added some BAC water to get free shipping):

Reconstitution and Filtering all-in-one Starter Kit — Peptide Test

Our Reconstitution and Filtering Starter Kit provides everything you need to safely reconstitute and filter water-soluble peptides. This kit includes all the components demonstrated in our instructional videos, ensuring a smooth and aseptic process. ️ - Click Here For Written and Video Instruct

www.peptidetest.com

 

[Sera]

I ordered one of these filter kits just to get started (added some BAC water to get free shipping):

Reconstitution and Filtering all-in-one Starter Kit — Peptide Test

Our Reconstitution and Filtering Starter Kit provides everything you need to safely reconstitute and filter water-soluble peptides. This kit includes all the components demonstrated in our instructional videos, ensuring a smooth and aseptic process. ️ - Click Here For Written and Video Instruct

www.peptidetest.com

I ended up going for the linked cobetter filters in the end.

 

[RandGuy]

Is it fair to say that all the 13mm PES sterile 0.22 µm hydrophilic syringe filters on the market are equivalent for our purposes (i.e., brand not important)?

(I guess volume remaining in filter may vary, dependent on prefilter.)

And what are the critical specs for the destination vials we should be using (lots of choices out there for under $2/vial, but not clear what we should be looking for). Thx

 

[Ateam2023]

Is it fair to say that all the 13mm PES sterile 0.22 µm hydrophilic syringe filters on the market are equivalent for our purposes (i.e., brand not important)?

(I guess volume remaining in filter may vary, dependent on prefilter.)

And what are the critical specs for the destination vials we should be using (lots of choices out there for under $2/vial, but not clear what we should be looking for). Thx

This has been discussed AT LENGTH several times,, are you even trying to use the "search function "?

 

[RandGuy]

Well, past posts don't answer either of my questions: what to make of filters other than Pall/Tisch/Cobetter, or what exactly are the criteria for choosing destination vials for the filtrate that don't accelerate aggregate formation (i.e., criteria for both that can be used to make smart choices on places like Amazon). But not a big deal, and thanks to everyone who has contributed to the discussion.

Anyway, am going with readily available Tisch filters (0.02 mL holdup volume) from domestic source, and trying Ghoul's suggestion of storing the reconstituted fluid in the syringe with filter kept attached, dispensing doses from that as needed (like a pen), eliminating need for a second vial.

 

[Ghoul]

Well, past posts don't answer either of my questions: what to make of filters other than Pall/Tisch/Cobetter, or what exactly are the criteria for choosing destination vials for the filtrate that don't accelerate aggregate formation (i.e., criteria for both that can be used to make smart choices on places like Amazon). But not a big deal, and thanks to everyone who has contributed to the discussion.

Anyway, am going with readily available Tisch filters (0.02 mL holdup volume) from domestic source, and trying Ghoul's suggestion of storing the reconstituted fluid in the syringe with filter kept attached, dispensing doses from that as needed (like a pen), eliminating need for a second vial.

Vials are easy. ultra spec.

Buy Ultra Spec Sterile 10ml-20mm Clear Sealed Glass Vials

In order to receive vials in sealed shrink wrap boxes, please order in multiples of 25

www.medical-and-lab-supplies.com

Ultra Spec Sterile 5ml (20mm neck) Clear Sealed Glass Vials

In order to receive vials in sealed shrink wrap boxes, please order in multiples of 25

www.medical-and-lab-supplies.com

Buy Ultra Spec Sterile 2ml Clear Sealed Glass Vials at wholesale prices

In order to receive vials in sealed shrink wrap boxes, please order in multiples of 25

www.medical-and-lab-supplies.com

 

[RandGuy]

Thx. Was just looking for more than a brand name so that I might choose less expensive or smaller volume vials. But probably more sensible to simply buy the Ultra Spec than try to figure out if other vials would also minimize aggregation.

 

[Ghoul]

Thx. Was just looking for more than a brand name so that I might choose less expensive or smaller volume vials. But probably more sensible to simply buy the Ultra Spec than try to figure out if other vials would also minimize aggregation.

It's not going to get any cheaper than $1.50-$2 for vials certified to meet USP/FDA standards for sterility and most importantly, free of particulates.

 

[CrazyDaisy]

Just commenting to say thank you for the info provided and answers to the extra questions posed. I just ordered the vials Ghoul recommended as well as supplies from Peptide Test above (and their video is helpful for newbies, too).

 

[RandGuy]

I investigated the issue of dead space/volume (fluid left in syringe/filter/needle) after injection. On inlet side, it appears dead space can be largely eliminated with use of a reduced dead space luer lock syringe (the ones with the extended plunger), but on outlet side of filter could not find a low dead space needle of reasonable price or dimension for direct injection (i.e., to treat syringe+filter like a pen). So will try back-filling multiple (low dead space) insulin syringes with the filtrate (then refrigerated), to be used as needed within a few days, eliminating need for the 2nd vial. (A variation of Ghoul's suggestion.)

 

[Ateam2023]

I investigated the issue of dead space/volume (fluid left in syringe/filter/needle) after injection. On inlet side, it appears dead space can be largely eliminated with use of a reduced dead space luer lock syringe (the ones with the extended plunger), but on outlet side of filter could not find a low dead space needle of reasonable price or dimension for direct injection (i.e., to treat syringe+filter like a pen). So will try back-filling multiple (low dead space) insulin syringes with the filtrate (then refrigerated), to be used as needed within a few days, eliminating need for the 2nd vial. (A variation of Ghoul's suggestion.)

backfilling is where your "freshly filtered" solution is going to "possibly" pick up contamination,, Its such a crapshoot honestly, just like injecting solutions that havent been filtered,,

 

[Ghoul]

backfilling is where your "freshly filtered" solution is going to "possibly" pick up contamination,, Its such a crapshoot honestly, just like injecting solutions that havent been filtered,,

Not quite. It's not ideal in terms of potential bacterial contamination from the few seconds of the plunger side being exposed, but filtering is still removing particulates, aggregates, and preexisting bacteria.

In terms of risks of contamination from exposure to air, consider the ampule. The most common form of packaging worldwide of single dose injectables.



This exposes the injectable to a much larger area and for longer than backfilling an insulin syringe.

There's also the process of unwrapping a luer lock syringes and needles. Each of those have open ends exposed to non-sterile air until they're connected.



Again, not ideal, but considering these are both standard procedures performed in high hazard infectious environments like hospitals, I can only conclude the risk of bacterial contamination from small aperture openings must be very low.

 

[Ateam2023]

Not quite. It's not ideal in terms of potential bacterial contamination from the few seconds of the plunger side being exposed, but filtering is still removing particulates, aggregates, and preexisting bacteria.

In terms of risks of contamination from exposure to air, consider the ampule. The most common form of packaging worldwide of single dose injectables.

View attachment 324185

This exposes the injectable to a much larger area and for longer than backfilling an insulin syringe.

There's also the process of unwrapping a luer lock syringes and needles. Each of those have open ends exposed to non-sterile air until they're connected.

View attachment 324188

Again, not ideal, but considering these are both standard procedures performed in high hazard infectious environments like hospitals, I can only conclude the risk of bacterial contamination from small aperture openings must be very low.

i wasn't talking ampoules, i was referring to the process of backfilling an insulin syringe , albeit filtered , theres STILL a risk, not discounting the practices used in "hospital settings ", but just admit it @Ghoul "THERE IS A RISK" say it !! :---)

 

[Sampei]

i wasn't talking ampoules, i was referring to the process of backfilling an insulin syringe , albeit filtered , theres STILL a risk, not discounting the practices used in "hospital settings ", but just admit it @Ghoul "THERE IS A RISK" say it !! :---)

Backfilling has never been used in any hospital settings. There is a big difference between opening up a sterile ampoule and drawing with a filtered needle (that's what is used in a hospital to draw from ampoule) Vs backfilling an insuline syringe.

Most of the stories of abscess I have read started with: backfilling

 

[Ateam2023]

Backfilling has never been used in any hospital settings. There is a big difference between opening up a sterile ampoule and drawing with a filtered needle (that's what is used in a hospital to draw from ampoule) Vs backfilling an insuline syringe.

Most of the stories of abscess I have read started with: backfilling

ive done my share of backfilling and i just know there's that risk, Ghoul won't admit to anything that isn't "prim&proper" and filtered blah blah , risks are everywhere ,, Goddamn sun exposure is pretty dangerous,, lol

 

[Ghoul]

W

i wasn't talking ampoules, i was referring to the process of backfilling an insulin syringe , albeit filtered , theres STILL a risk, not discounting the practices used in "hospital settings ", but just admit it @Ghoul "THERE IS A RISK" say it !! :---)

Did you miss this or are you just getting off on pretending I said there's NO risk?



Jano says 1 in 20 peptides tested for sterility are contaminated with enough bacteria to fail. That's very high. What do you recommend users do to handle that?

Post in thread 'Readalots Enhanced Testing'

Dec 28, 2024

Failure rates for endotoxin and tamc+tymc the way we do it are indeed quite low, roughly 5%.

It's mostly peptides failing, though.

• janoshik

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