August 28, 2007
By Sam Massoni, Dr. Rangaraju Naramishachar and Dr. TK Prakasha
Synthetic peptides are an excellent tool in biopharmaceutical research. Their vast potential is best realized by following a few simple guidelines for peptide design, use, handling and storage.
When selecting a peptide sequence, be mindful that design for manufacturability is an important factor since some sequences can cause production delay or failure. When possible, follow these guidelines to aid in synthesis success.
Avoid problematic amino acid locations inside the sequence:
- A Glutamine (Q) residue at the N-terminus of the peptide may
undergo spontaneous transformation into a pyroGlutamic
residue. To prevent this transformation, acetylate the
N-terminus, add another amino acid N-terminally to the Q, or
remove the Q altogether.
- Cysteine (C) and Proline (P) have proven to be problematic
when located at the C-terminus of the peptide. To prevent
issues, amidate the C-terminus, add another amino acid
C-terminally to the C or P, or, eliminate the C or P altogether.
- An Aspartic Acid (D) residue located anywhere in the peptide
can spontaneously transform into an Aspartamide residue
regardless of its location in a sequence.
Watch out for certain amino acid combinations:
Aspartic Acid/Glycine (D/G) pairings or numerous Threonine (T), Cysteine (C), Leucine (L), Valine (V) or Isoleucine (I) residues in a row can be troublesome. Where possible, avoid such combinations in sequence selection in order to position a peptide for production success.
Recreating the Natural Environment
Peptides are created to mimic proteins or the cleavage products of proteins. When proteins are cleaved in vivo, they have naturally occurring free unprotected termini. Therefore blocking the termini is not necessary for in vivo cleavage. However, when the sequence is not a known cleavage product, blocking the termini is necessary in order to mimic the peptide bonds normally found in the parent sequence.
Use the following rules:
- If the sequence is C-terminal, block the N-terminus by acetylation
- If the sequence is N-terminal, block the C-terminus by amidation
- If the sequence is internal, block both ends with acetylation and amidation
Verifying Peptide Quality
Since production and quality control (“QC”) techniques vary by peptide producer, always verify the peptide quality prior to using the peptide. At a minimum, the QC check should include a detailed review of the quality documentation that accompanied the peptide. There are three key documents to review.
Mass Spectrometry – The mass spec confirms the peptide identity by reporting the molecular weight of the sample. The
resulting data should be within 0.1% Daltons of the theoretical molecular weight.
HPLC – The HPLC trace reports peptide purity. It is important to make certain that the HPLC data includes gradient time and percentage. A gradient faster than 2% per minute may mask impurities, making the peptide appear more pure than it actually is.
Certificate of Analysis – A certificate of analysis should detail the raw materials used (including corresponding manufacturers and
lot numbers), instruments, assay details and QC signatures. Proceeding with research without this level of traceability and
reproducibility is not recommended.
Selecting Target Purity
Selecting the appropriate peptide purity is assay dependent. The following are some basic research-driven guidelines:
|Application||Non-sensitive screening assays||Immunogen grade||Receptor/ligand, bio-assay or cell studies||Structural Studies|
|Recommended Purity||Crude or >75%|
Reconstituting a Peptide
Reconstituting a peptide can pose challenges, but proper planning can eliminate common problems.
- Bring frozen or refrigerated peptides to room temperature in a desiccated chamber to avoid water absorption.
- Always begin by reconstituting a small amount of peptide before committing the entire lot.
- Use sterile water or sterile filtration. If there are any Methionine (M), Cysteine (C), or Tryptophan (W) residues, use oxygen free solvents to prevent oxidation. Avoid reconstituting a peptide in a buffer, like PBS, since salts hinder solubility.
- A solubilized peptide is completely clear. No flecks or cloudiness should be present.
- Should the peptide not go into solution, re-lyophilize the peptide and begin again or centrifuge or filter the peptide to remove insoluble particles.
|Amino Acid Characteristics||Recommended Solvent|
|Hydrophilic residues (KRHDEPN)||H2O*|
|Hydrophobic residues (AVLIMFWP)||Low solubility in aqueous solvents; are soluble in|
organic solvents (DMF, DMSO, TFA, Acetonitrile)
*If the peptide does not go into solution completely, calculate the pI of the peptide and add either 0.1N acetic acid or 0.1N ammonium acetate to adjust pH away from the pI until solubilized. If the peptide still does not go into solution, try organic solvents such as DMSO, Acetonitrile or DMF.
Properly Storing a Peptide
Proper storage of a peptide can prevent bacterial degradation, secondary structure formation, oxidation and other potential degradation for several years. Peptides are most stable in their lyophilized form at -20° C or colder in a sealed container containing desiccant. If peptide must be stored in solution, ensure pH is in the 3-6 range and then aliquot peptide into usable sizes to prevent damage from multiple freeze/thaw cycles. Cysteine (C), Methionine (M), Tryptophan (W), Asparagine (N) and Glutamine (Q) are most sensitive to degradation in solution.
|Long-term Storage||3 months|
to 5 years
|Lyophilized powder, frozen and|
desiccated, -20° C or colder
|Frozen liquid (-20° C) or|
refrigerated lyophilized powder
In summary, follow the design and use guidelines detailed above to harness the potential of peptides in biopharmaceutical research.
About the Authors
Sam Massoni, senior scientist and founder of New England
Peptide, is a career peptide chemist who continues to lead the
industry in new directions.
Rangaraju Naramishachar PhD, a senior scientist at New
England Peptide, is a 25 year veteran of the peptide
industry who has led production teams and trained peptide
TK Prakasha PhD, After focusing the early portion of his career in
R&D at DuPont, TK took on oversight of new product development and
business development at Borregaard Synthesis.
He currently drives business development at New England
NOTE: All production-related advice is based on FMOC solid phase peptide synthesis production methods.