Abstract
Advances in recombinant DNA technology have opened up new possibilities of exploiting toxic proteins for therapeutic purposes. Bringing forth these protein toxins from the bench to the bedside strongly depends on the availability of production methods that are reproducible, scalable and comply with good manufacturing practice (GMP). The type I ribosome-inhibiting protein, gelonin, has great potential as an anticancer drug, but is sequestrated in endosomes and lysosomes. This can be overcome by combination with photochemical internalization (PCI), a method for endosomal drug release. The combination of gelonin-based drugs and PCI represents a tumor-targeted therapy with high precision and efficiency. The aim of this study was to produce recombinant gelonin (rGel) at high purity and quantity using an automated liquid chromatography system. The expression and purification process was documented as highly efficient (4.4 mg gelonin per litre induced culture) and reproducible with minimal loss of target protein (~50% overall yield compared to after initial immobilized metal affinity chromatography (IMAC)). The endotoxin level of 0.05–0.09 EU/mg was compatible with current standards for parenteral drug administration. The automated system provided a consistent output with minimal human intervention and close monitoring of each purification step enabled optimization of both yield and purity of the product. rGel was shown to have equivalent biological activity and cytotoxicity, both with and without PCI-mediated delivery, as rGelref produced without an automated system. This study presents a highly refined and automated manufacturing procedure for recombinant gelonin at a quantity and quality sufficient for preclinical evaluation. The methods established in this report are in compliance with high quality standards and compose a solid platform for preclinical development of gelonin-based drugs.
Highlights
The development of protein toxins as cancer therapeutics is still in its infancy
Key Contribution: This study presents a highly refined, automated method for production of gelonin at a quantity and quality sufficient for extensive preclinical evaluation
The DNA fragment of recombinant gelonin (rGel) was synthesized with BspHI and XhoI restriction sites introduced in fragment of rGelThe wasrGel synthesized with sites introduced in the 5′The andDNA
Summary
The development of protein toxins as cancer therapeutics is still in its infancy. Building the translational bridge from basic research to clinical drug application is a complex and multifaceted process. One of the main identified reasons for high failure rates and discontinuation of clinical drug development is the inability to reproduce preclinical data [2]. Toxins 2020, 12, 519 in small scale in the research lab are not necessarily translatable to industrial scale and biomedical companies invest both time and money in optimizing procedures that may not be replicable. A study published in 2015 estimates that, in the United States alone, approximately 28 billion USD are invested annually on preclinical research that is not reproducible [3]. To support clinical drug translation, there is a need for standardized and scalable methods that minimize sources of irreproducibility
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