Jecho Laboratories Inc.

Frederick, MD, United States

Jecho Laboratories Inc.

Frederick, MD, United States
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Chen H.,Shanghai JiaoTong University | Li N.,Shanghai JiaoTong University | Xie Y.,Jecho Laboratories Inc. | Jiang H.,Jecho Laboratories Inc. | And 12 more authors.
Applied Microbiology and Biotechnology | Year: 2017

It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems. © 2017 Springer-Verlag Berlin Heidelberg


Sun T.,Shanghai JiaoTong University | Li C.,Shanghai JiaoTong University | Han L.,Shanghai JiaoTong University | Jiang H.,Jecho Laboratories Inc. | And 6 more authors.
Engineering in Life Sciences | Year: 2015

We report the adaptation of the new CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system to disrupt the gene encoding fucosyltransferase 8 (FUT8), an α1,6-fucosyltransferase that directs fucose addition to derived antibody Fc region asparagine 297, in Chinese hamster ovary (CHO) cells. Compared to previously reported homologous recombination or zinc-finger nucleases (ZFNs) applications in CHO cells, CRISPR/Cas9 demonstrated higher targeting efficiency and easier customization. FUT8 disruptive clones (FUT8-/-) were obtained within 3 weeks at indel frequencies ranging from 9 to 25%, which could be enhanced to 52% with Lens culinaris agglutinin (LCA) selection. Based on the lectin blot method, the derived FUT8-/- clone had the ability to produce defucosylated therapeutic mAb with no detrimental effects on cell growth, viability, or product quality. The clone had the potential of industrial application for therapeutic antibodies manufacturing. We have demonstrated functionally that a gene related to product synthesis could be mutated using CRISPR/Cas9 technology, and consequently the glycan profile of expressed mAb was alternated. We believe that with its robustness and effectiveness, CRISPR/Cas9 can be widely applicable in cell line development leading to higher productivity and better quality of mAbs and other biological therapeutics. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Shi S.,Shanghai JiaoTong University | Chen H.,Shanghai JiaoTong University | Jiang H.,Jecho Laboratories Inc. | Xie Y.,Jecho Laboratories Inc. | And 10 more authors.
Applied Microbiology and Biotechnology | Year: 2016

Soluble expression of recombinant therapeutic proteins in Escherichia coli (E. coli) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic–intein has been constructed for the soluble expression and purification of a recombinant cytokine, human interleukin-15 (IL-15). We screened several solubilizing tags fused with the self-cleavable Mycobacterium tuberculosis recA mini-intein ∆I-CM and demonstrated that Zbasic tag can significantly improve the solubility of the product with correspondent to the intein activity. The fusion protein “Zbasic–∆I-CM–IL-15” was expressed with high solubility and easily enriched by the cost-effective cation-exchange chromatography. The self-cleavage of the fusion tag Zbasic–∆I-CM was then induced by a pH shift, with an activation energy of 7.48 kcal/mol. The mature IL-15 with natural N-terminus was released and further purified by hydrophobic interaction and anion-exchange chromatography. High-resolution reverse-phase high-performance liquid chromatography and mass spectrometry analysis confirmed that the product was of high purity and correct mass. With a CTLL-2 cell proliferation-based assay, the EC50 was evaluated to be of about 0.126 ng/mL, similar to the product in clinical trials. By avoiding the time-consuming denaturing-refolding steps in previously reported processes, the current method is efficient and cost-effective. The novel tag Zbasic–∆I-CM can be potentially applied to large-scale manufacturing of recombinant human cytokines as well as other mammalian-sourced proteins in E. coli. © 2016 Springer-Verlag Berlin Heidelberg


PubMed | Jecho Laboratories Inc. and Shanghai JiaoTong University
Type: Journal Article | Journal: Applied microbiology and biotechnology | Year: 2016

Soluble expression of recombinant therapeutic proteins in Escherichia coli (E. coli) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic-intein has been constructed for the soluble expression and purification of a recombinant cytokine, human interleukin-15 (IL-15). We screened several solubilizing tags fused with the self-cleavable Mycobacterium tuberculosis recA mini-intein I-CM and demonstrated that Zbasic tag can significantly improve the solubility of the product with correspondent to the intein activity. The fusion protein Zbasic-I-CM-IL-15 was expressed with high solubility and easily enriched by the cost-effective cation-exchange chromatography. The self-cleavage of the fusion tag Zbasic-I-CM was then induced by a pH shift, with an activation energy of 7.48kcal/mol. The mature IL-15 with natural N-terminus was released and further purified by hydrophobic interaction and anion-exchange chromatography. High-resolution reverse-phase high-performance liquid chromatography and mass spectrometry analysis confirmed that the product was of high purity and correct mass. With a CTLL-2 cell proliferation-based assay, the EC

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