Development of a platform process for the production and purification of single-domain antibodies

LAURA E. CROWELL; CHAZ GOODWINE; SOSA HOLT, CARLA; LUCIA ROCHA; CELINA VEGA; SERGIO A. RODRIGUEZ; NEIL C. DALVIE; MARY K. TRACEY; MARIANA PUNTEL; ANDRES WIGDOROVITZ; VIVIANA PARREÑO; KERRY R. LOVE; STEVEN M. CRAMER; J. CHRISTOPHER LOVE

Biotechnology and Bioengineering

John Wiley & Sons, Inc.

Lugar: New Jersey; Año: 2020

Single-domain antibodies (sdAbs) offer the affinity and therapeutic value ofconventional antibodies, with increased stability and solubility. Unlike conventionalantibodies, however, sdAbs do not benefit from a platform manufacturing process.While successful production of a variety of sdAbs has been shown in numerous hosts,purification methods are often molecule specific or require affinity tags, whichgenerally cannot be used in clinical manufacturing due to regulatory concerns. Here,we have developed a broadly applicable production and purification process forsingle-domain antibodies in Komagataella phaffii (Pichia pastoris) and demonstratedthe production of eight different sdAbs at a quality appropriate for nonclinical studies.We developed a two-step, integrated purification process without the use of affinityresins and showed that modification of a single process parameter, pH of the bridgingbuffer, was required for the successful purification of a variety of sdAbs. Further, wedetermined that this parameter can be predicted based only on the biophysicalcharacteristics of the target molecule. Using these methods, we produced nonclinicalquality sdAbs as few as five weeks after identifying the product sequence. Nonclinicalstudies of three different sdAbs showed that molecules produced using our platformprocess conferred protection against viral shedding of rotavirus or H1N1 influenzaand were equivalent to similar molecules produced in E. coli and purified usingaffinity tags.