Contributions
Abstract: EP887
Type: E-Poster Presentation
Session title: Lymphoma Biology & Translational Research
Background
Recombinant immunotoxins (rIT) are fusion proteins of a targeting moiety and pseudomonas exotoxin (PE). They arrest protein synthesis in eukaryotes by ADP-ribosylation of elongation factor 2 (eEF2) leading to target cell death. The CD22 targeted immunotoxin Moxetumomab pasudotox (Moxe) was approved for the treatment of relapsed/refractory hairy cell leukemia. Because immunotoxins stop eukaryotic protein expression, they are expressed in E. coli. However, proteins expressed in E. coli are contaminated by endotoxin (also called lipopolysaccharides (LPS)). LPS binds to toll-like receptors, inducing up to life-threatening systemic inflammation in mammals. Therefore, FDA-approved LPS-limit for therapeutics as well as for substances used in immunological studies in animals are as low as 0.05 EU/mg.
Aims
We aimed to establish a broadly applicable protein purification method to reduce LPS below FDA approved limits without altering stability or function of the protein.
Methods
Immunotoxins were expressed in E. coli, refolded, and purified using standard protocol by three column chromatography. To reduce LPS, an on column Triton X-114 wash, amine based spin columns, or the combination of the two was tested. LPS levels were measured by LAL-assay, protein quality control was done by SDS-PAGE analysis. Immunotoxins were tested extensively in vitro for ADP-ribosylation, cytotoxicity, thermal stability, and binding affinity. In vivo efficacy was determined in a systemic xenograft model.
Results
Moxe produced by standard method was contaminated by 2.7 EU/mg LPS. The on column Triton X-114 wash did not reduce LPS contamination by itself and spin-columns reduced LPS to 0.40 EU/mg. When combined, LPS was reduced by 120-fold to 0.02 EU/mg compared to standard method. Immunotoxins purified by the combination were purer by 9% which we found to be a result of the Triton X-114 on-column wash. Furthermore, in vitro cytotoxicity was enhanced up to 2.4-fold which was not explained by increased enzymatic ADP-ribosylation activity or improved protein stability, but by an enhanced binding affinity to CD22 on the cell surface. LPS-reduced protein maintained in vivo efficacy. Additionally, LPS was reduced below FDA-limits for other antibody formats (dsFv and Fab), for distinct PE variants and for antibodies against distinct targets.
Conclusion
Only Triton X-114 on-column wash combined with amine-residue-based spin columns reliably reduced LPS contamination below FDA-approved limits. Resulting immunotoxins were purer and more potent. With some modifications, the method may be applicable for any chromatography-based purification processes and may be run entirely on column.
Keyword(s): Antibody, Immunotoxin, LPS, Targeted therapy
Abstract: EP887
Type: E-Poster Presentation
Session title: Lymphoma Biology & Translational Research
Background
Recombinant immunotoxins (rIT) are fusion proteins of a targeting moiety and pseudomonas exotoxin (PE). They arrest protein synthesis in eukaryotes by ADP-ribosylation of elongation factor 2 (eEF2) leading to target cell death. The CD22 targeted immunotoxin Moxetumomab pasudotox (Moxe) was approved for the treatment of relapsed/refractory hairy cell leukemia. Because immunotoxins stop eukaryotic protein expression, they are expressed in E. coli. However, proteins expressed in E. coli are contaminated by endotoxin (also called lipopolysaccharides (LPS)). LPS binds to toll-like receptors, inducing up to life-threatening systemic inflammation in mammals. Therefore, FDA-approved LPS-limit for therapeutics as well as for substances used in immunological studies in animals are as low as 0.05 EU/mg.
Aims
We aimed to establish a broadly applicable protein purification method to reduce LPS below FDA approved limits without altering stability or function of the protein.
Methods
Immunotoxins were expressed in E. coli, refolded, and purified using standard protocol by three column chromatography. To reduce LPS, an on column Triton X-114 wash, amine based spin columns, or the combination of the two was tested. LPS levels were measured by LAL-assay, protein quality control was done by SDS-PAGE analysis. Immunotoxins were tested extensively in vitro for ADP-ribosylation, cytotoxicity, thermal stability, and binding affinity. In vivo efficacy was determined in a systemic xenograft model.
Results
Moxe produced by standard method was contaminated by 2.7 EU/mg LPS. The on column Triton X-114 wash did not reduce LPS contamination by itself and spin-columns reduced LPS to 0.40 EU/mg. When combined, LPS was reduced by 120-fold to 0.02 EU/mg compared to standard method. Immunotoxins purified by the combination were purer by 9% which we found to be a result of the Triton X-114 on-column wash. Furthermore, in vitro cytotoxicity was enhanced up to 2.4-fold which was not explained by increased enzymatic ADP-ribosylation activity or improved protein stability, but by an enhanced binding affinity to CD22 on the cell surface. LPS-reduced protein maintained in vivo efficacy. Additionally, LPS was reduced below FDA-limits for other antibody formats (dsFv and Fab), for distinct PE variants and for antibodies against distinct targets.
Conclusion
Only Triton X-114 on-column wash combined with amine-residue-based spin columns reliably reduced LPS contamination below FDA-approved limits. Resulting immunotoxins were purer and more potent. With some modifications, the method may be applicable for any chromatography-based purification processes and may be run entirely on column.
Keyword(s): Antibody, Immunotoxin, LPS, Targeted therapy