To filter or not filter?

[StonePny]

Are there certain peptides that you (the collective you) always filter vs. those you don't think necessary? Your glp's, Ipamorelin, CJC-1295? Are there historically more "dirty" ones?

 

[Calm Logic]

The users who are too unmotivated to filter all the time may not have the motivation to reply

I would assume every peptide being freeze-dried is basically equally at risk regarding sterility due to various sterilities in manufacturing, fillers used, etc:

In practice, for any peptide intended for sterile administration, sterile filtration is a non-negotiable step. The risk of introducing microbial contamination or particulate matter during handling outweighs any subtle differences in their inherent stability regarding aggregation.

But if sterility is less of a concern (which seems crazy to me, though the BAC water does help), then it is the GLP-1s that are more likely to need filtering due to aggregation:

• More Stable Peptide (Example: Oxytocin): Oxytocin is a relatively small cyclic nonapeptide (9 amino acids) containing a disulfide bridge, which can contribute to its structural stability. While it's still susceptible to degradation under certain conditions (like high temperatures or extreme pH), its inherent structure provides a degree of robustness compared to some larger linear peptides.
  
  
• Less Stable Peptide (Example: Growth Hormone-Releasing Hormone - GHRH): GHRH is a longer peptide (44 amino acids) known to be more susceptible to degradation. It can undergo oxidation, hydrolysis, and aggregation more readily than smaller, structurally constrained peptides. Therefore, the freeze-drying process and the choice of excipients become even more critical for maintaining its integrity.
  
  
• Peptide Sensitive to Oxidation (Example: Melanotan II): Melanotan II is a cyclic heptapeptide containing methionine, an amino acid that is particularly prone to oxidation. During freeze-drying and subsequent storage, even trace amounts of oxygen can lead to the formation of methionine sulfoxide, potentially affecting the peptide's efficacy.
  
  
• Peptide Prone to Aggregation (Example: Amyloid-beta fragments): Certain peptides, like fragments of amyloid-beta, have a high propensity to aggregate, especially under stress conditions. The freeze-drying process itself can sometimes induce or exacerbate aggregation. Careful selection of cryoprotectants and optimization of the freeze-drying cycle are crucial for these peptides.
  
  Glucagon-like peptide-1 (GLP-1) receptor agonists (e.g., Exenatide, Liraglutide, Semaglutide): These longer peptides, used for treating type 2 diabetes and obesity, can be prone to aggregation due to their size and specific amino acid sequences. Freeze-drying formulations often include excipients to minimize aggregation and maintain their soluble, monomeric form upon reconstitution.
  
  
• Peptide Sensitive to Hydrolysis (Example: Peptides with Aspartyl-Prolyl bonds): Peptides containing Aspartyl-Prolyl (Asp-Pro) sequences are known to be susceptible to acid-catalyzed hydrolysis and isomerization. The pH during the freeze-drying process and in the reconstituted solution needs to be carefully controlled to prevent degradation at these specific sites.
  
  Oxytocin (again, as an example of a cyclic peptide): While its cyclic structure provides some stability, the peptide bonds within oxytocin can still undergo hydrolysis over time, especially under non-optimal storage conditions. The rate of hydrolysis might be slower than in some linear peptides, but it's still a potential degradation pathway.

Filtering can definitely help in certain aspects of ensuring a "cleaner" peptide solution, but it's not a universal solution for all potential issues. Here's a breakdown of how filtering can help and what it might not address:

How Filtering Can Help:

• Removing Particulate Matter and Aggregates: Filters with appropriate pore sizes (typically 0.2 µm or larger for general particle removal, and even finer for sterilization) can physically remove aggregated peptides and other particulate impurities that might have formed during freeze-drying or reconstitution. This can lead to a visually cleaner and potentially safer solution, especially for injectable products.
  
  
• Sterilization: Using sterile filters with a pore size of 0.22 µm is a common method for removing bacteria and other microorganisms from a peptide solution after reconstitution. This is crucial for ensuring the sterility of the final product, especially if it's intended for in vivo use or cell culture.
  
  
• Clarification: Filtration can remove larger, insoluble components that might cloud the solution, resulting in a clearer and more aesthetically pleasing product.

What Filtering Might Not Address:

• Hydrolyzed Peptides (Degradation Products): Hydrolysis breaks the peptide down into smaller chemical fragments that are typically dissolved in the solution. Standard filters will not remove these smaller molecules, as they are below the pore size of the filter.
  
  
• Other Soluble Impurities: Filters won't remove other soluble impurities that might have been introduced during manufacturing (e.g., residual solvents, counterions from purification) or leached from the vial or stopper.
  
  
• Endotoxins: While filters can remove the bacteria that produce endotoxins, the endotoxin molecules themselves are relatively small and may pass through standard sterilization filters. Specific endotoxin removal filters are needed to address this issue.
  
  
• Viral Contamination:Standard 0.22 µm filters are generally effective for bacteria but may not reliably remove all viruses, which are much smaller. Filters with smaller pore sizes (e.g., ultrafiltration membranes) are required for virus removal.
  
  
• The Inherent Instability of the Peptide: Filtering doesn't change the fundamental chemical properties of the peptide that make it prone to aggregation or hydrolysis over time. While it can remove existing aggregates, it won't prevent future aggregation from occurring. Similarly, it won't stabilize a peptide that is susceptible to hydrolysis.

Key Considerations for Filtering Peptides:

• Filter Pore Size: The choice of pore size depends on the intended purpose. Sterile filtration requires 0.22 µm filters, while larger pore sizes can be used for clarification or removing larger aggregates.
• Filter Material:Some filter materials can adsorb peptides, leading to a loss of product. Low protein-binding membranes (e.g., PVDF, PES) are often preferred for peptide solutions.
  
  
  
• Sterility of the Filter: If sterilization is the goal, the filter itself must be sterile.
• Pre-wetting:Some filters require pre-wetting with a suitable solvent before use to ensure proper flow and prevent air bubbles.

In conclusion, filtering is a valuable tool for removing particulate matter and sterilizing peptide solutions. However, it's not a solution for all types of impurities or the inherent instability of the peptide molecule itself. A comprehensive approach to ensuring a high-quality freeze-dried peptide product involves stringent manufacturing processes, appropriate formulation, proper storage, and potentially filtration as one step in the process.

Also:

Every "purity" test you see of UGL peptides is conducted AFTER being filtered with a .22um syringe filter. Contaminants larger than that, which are common, are removed first.

Unless you're filtering first, you're not injecting what you see in the test result, but the "raw" version with some unknown amount of contamination, consisting of glass delimitation shards, rubber particulates, bacteria, peptide aggregates, etc.