vivitide specializes in the production of difficult peptides by incorporating many methods to overcome difficulties in the manufacturing process. While we are known for being able to produce the most difficult peptides, it is always best to avoid known areas of concern when possible.  An overly uncooperative sequence, even when able to be produced, can cause production delays, increased manufacturing costs and difficulty in future scale up. Our expert chemists specialize in sequence design that optimizes stability, solubility, manufacturability of scale up, while keeping the core active region of a peptide intact.

While the below tips are meant to help solubility, stability and future scale up, we understand changing a peptides amino acid composition isn’t always possible. In these cases, our expert chemists will optimize a custom protocol to ensure a timely, cost-effective delivery, even when challenges present themselves.

Peptide solubility- Avoiding an overly hydrophobic sequence will allow higher yields, better impurity separation, decreased timelines and lowers costs for future scale up. A rule of thumb is to try to have less than 40% hydrophobic residues (A,V,L,I,M,F,W,P) in your peptide while also incorporating as many hydrophilic/charged residues (K,R,H,D,E) as the design will allow.  While most problematic solubility can be overcome with the use of alternative solvents and sequence specific pH adjustments, it can cause low yields and inconsistency with future workup while in the hands of the scientist performing experimental lab work.

Pyroglutamate formation- A Glutamine (Q) residue at the N-terminus (H2N-QXXXX) of the peptide may undergo spontaneous transformation into a pyroglutamic (pGlu) residue causing a mixture pGlu and Glutamine. We can provide as  a mixture or to prevent such behavior, acetylate the N terminus (Ac-QXXXX) or incorporate a pGlu residue in place of the Glutamine.

Aspartimide formation- Aspartimide formation is a side reaction caused by repeated exposure to piperidine or other bases during fmoc SPPS. This can be limited by the use of alternative protecting groups, the use of dimer amino acids and adding HOBt to the piperidine deprotection solution. Aspartic Acid, Glycine (DG) is the most prone to cause aspartimide formation. Limiting the number of DG combos is a peptide can greatly simplify the manufacturing process especially when near the c-terminus of a peptide. While the DG combo is the most prone to aspartimide formation, there are several other combos that including DS and DA.

Beta branched amino acids- Avoid long continuous strings of beta branched amino acids Isoleucine (I), Valine (V) and Threonine (T). These are structurally hindered and when multiple are coupled together, they can become increasingly challenging. If this is unavoidable, we can employ more aggressive couplings/deprotections, more potent activation methods, alternative resins, microwave couplings and many other sequence specific optimizations to overcome.

Diketopiperizine formation- C-term Proline (P-OH) should be amidated when possible. Proline acid can undergo diketopiperizine formation causing low or no yield from the synthesis of the peptide. This can be avoided by the use of  2-chlorotrityl chloride resin but this resin is less flexible when it comes to the use of heat and other optimizations that are needed if the sequence has further challenging regions.

Beta piperidinyl alanine formation- C-terminal Cysteine (XXXXC-OH) can cause Beta piperidinyl alanine formation. This results in a modification causing the breakdown of cysteine into, 3-(1-piperidinyl) alanine adduct. This can be avoided by the use of alternative resins or by amidating the sequence (XXXXC-Amide).

Please reach out to to speak to one of our expert chemists about peptide design or to discuss the many optimizations we employ to deliver even the most challenging of projects.