A new universal RP-HPLC column for peptide purification, and the use of novel flow-through media for the removal of acidic ion pairing agents and peptide free base formation are reported. The use of these two products, with SOPs, can substantially increase the throughput of peptide manufacture.
Getting a biotherapeutic synthetic peptide from the candidate identification stage through to production takes approximately 10 years. Initially small quantities, μg to g of many hundreds or thousands of peptides, 5 to 60 amino acids in length, are produced. To maximize throughput, standard methods of synthesis and purification are used. Reliable supplies of high quality synthesis supports, and reproducible, universally applicable columns for purification are needed to minimize failure at the synthesis stage, and eliminate method development at the purification step. A new universal purification column, VariTide RPC, and media, VariPure IPE (Ion Pair Extraction), for the removal of TFA or conversion of the peptide to the free base after purification, are tools for achieving this goal.
The VariTide RPC, 250 x 4.6 mm ID column, was used to chromatograph a mixture of peptides and small protein sequences, using 0.1% TFA in acetonitrile, and then to determine the optimum pH for purification by screening using: 0.1% TFA; 20 mM ammonium acetate, pH 5.5; 20 mM ammonium carbonate, pH 9.5; and 10 mM ammonium hydroxide, pH 10.4 with a linear gradient from 5 to 50% acetonitrile. To determine the capacity of the VariPure IPE media needed to remove TFA from the HPLC fractions, volumes of 0.1% TFA in 10–50% acetonitrile were passed though the media and the pH of the eluate measured. The media was considered to be fully exchanged, with the maximum amount of TFA removed, when the pH of the eluate was that of the 0.1% TFA solution.
To be universally applicable for the range of synthetic peptides, the pore size must be large enough to allow good mass transfer of the largest peptide but small enough to have good capacity. The separation of the peptide or protein mixture showed no band broadening compared to a 300Å size material, indicating good mass transfer over the required peptide size range. Figure 1 clearly shows the benefit of screening synthetic peptide resolution over a range of eluent pHs. An additional failure sequence from the solid phase synthesis is evident at high pH. From the screening runs, the optimum pH and gradient for the purification can be determined. The VariPure IPE media exchanges the TFA in the presence of the acetonitrile, with a minimum of 87% of theoretical capacity being used. When working at the recommended two-fold excess, 100 mg of the media exchanged 5 mL of 0.1% TFA solution–fraction from a preparative column.
A single column, VariTide RPC, can be used for synthetic peptide purification under acidic, neutral, and basic conditions. Peptides ranging in size from 5 to 60 amino acid residues are purified using one column, VariTide RPC, from μg to g scale. VariPure IPE media (100 mg) will free base 0.1 mmole of peptide and exchange 5 mL of 0.1% TFA solution.
Authors: Dr. Linda L. Lloyd, Dr. Paul A. Boguszewski
Polymer Laboratories, Varian, Inc.
Amherst Fields Research Park
160 Old Farm Road
Amherst, MA 01002