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

Single-domain antibodies (sdAbs) offer the affinity and therapeutic value of conventional antibodies, with increased stability and solubility. Unlike conventional antibodies, 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, which generally cannot be used in clinical manufacturing due to regulatory concerns. Here, we have developed a broadly applicable production and purification process for sdAbs in Komagataella phaffii (Pichia pastoris) and demonstrated the production of eight different sdAbs at a quality appropriate for nonclinical studies. We developed a two-step, integrated purification process without the use of affinity resins and showed that modification of a single process parameter, pH of the bridging buffer, was required for the successful purification of a variety of sdAbs. Further, we determined that this parameter can be predicted based only on the biophysical characteristics of the target molecule. Using these methods, we produced nonclinical quality sdAbs as few as 5 weeks after identifying the product sequence. Nonclinical studies of three different sdAbs showed that molecules produced using our platform process conferred protection against viral shedding of rotavirus or H1N1 influenza and were equivalent to similar molecules produced in Escherichia coli and purified using affinity tags.

Use of subcutaneous transponders to monitor body temperature in laboratory rats.

Implantable radiofrequency transponders may be adequate for the characterization of hazardous chemicals targeting body temperature control in experimental animals when colonic probes and automated monitoring systems based on intraperitoneal transmitters are not available, installable or applicable for any reason. In this work, we summarize a series of experiments showing the implantation protocol and utility of rice-grain size transponders to monitor subcutaneous temperature (Tsc) after exposure to pharmacological or toxicological treatments targeting body temperature control in laboratory rats. In addition, to explore the responsiveness of this thermometric system, the influence of physiological activity on Tsc readings was examined by monitoring Tsc after a motor exercise in a RotaRod system. Moreover, we characterized the effects of acute oral administration of the pyrethroid insecticide permethrin (PRM) in corn oil (1 mL/kg) on Tsc. PRM has been previously reported to cause dose-related increases in core temperature after administering oral doses ≥75 mg/kg, with peak effects at 2-4 h in adult rats. We monitored Tsc at 30 min intervals over a 4 h period after exposure to PRM (40-160 mg/kg). PRM caused a moderate increase in Tsc starting at ~3.5 h. Overall, Tsc assays showed minimal animal stress (if any) and rapid animal recovery from transponder implantation, simplicity to collect data, convenient testing room space requirements, and a competitive global cost per animal examined. However, various experimental factors may greatly influence the variability within and between individuals, some of which can be controlled by carefully setting up experimental conditions.