Historically peptide synthesis has been limited in the sequence length of compounds generated due to multiple factors, including low synthetic yields, crude mixture complexity, and purification difficulty of closely eluting, sequence similar contaminants. Recent advances in both the synthetic and analytical chemistry spaces have pushed this length beyond what was previously thought possible. While the syntheses improve, the purification stage has struggled to keep pace with growing synthetic capacity. This gap is magnified by the growth of AI based research pipelines, as the number of sequences to be transferred from in-silico to in-vitro for testing has increased dramatically. Improvements in automation have allowed purification teams to keep pace with synthetic teams for short peptides (usually <30 amino acids), but as peptide length increases so does purification complexity. Here we describe the adaptation of a high-throughput small molecule library purification methodology to peptide purification, moving from standard libraries of 10-12mers to beyond 70 amino acids in length while maintaining the same throughput as previous small molecule studies. This method leverages advancements in mass spectrometry, resin chemistry, and automation to enable the production of higher quality libraries of larger peptide molecules without sacrificing speed.
