Bench to Bedside: Stability Studies of GMP Produced Trastuzumab-TCMC in Support of a Clinical Trial

Abstract

The first-in-human phase 1 clinical radioimmunotherapy (RIT) trial with 212Pb-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoylmethyl)-cyclododecane-trastuzumab (212Pb-TCMC-trastuzumab) was completed in October 2014 as a joint effort at the University of Alabama (UAB) and the University of California San Diego Moores Cancer Center. The preliminary reports indicate that after five dose-levels of intraperitoneally administered 212Pb-TCMC-trastuzumab, patients with carcinomatosis experienced minimal agent-related toxicity. This report presents the data accumulated to date on the stability of the clinical grade, produced according to current good manufacturing practices (cGMP), TCMC-trastuzumab conducted in support of that clinical trial. Of the eleven tests performed with the cGMP TCMC-trastuzumab all but one remained within specifications throughout the 5 year testing period. The protein concentration varied by 0.01 mg/mL at 48 months. Two other assays, ion-exchange high performance liquid chromatography (IEX-HPLC) and a competitive radioimmunoassay (RIA) indicated that the cGMP TCMC-trastuzumab integrity may be changing, although the change thus far is within specifications. Subsequent stability testing will confirm if a trend has truly developed. The cGMP TCMC-trastuzumab was also evaluated for tolerance to higher temperatures and the potential of storage at −80 °C. The immunoconjugate proved stable when subjected to the lower temperatures and to multiple freeze-thaw cycles. The size exclusion (SE) HPLC analysis of the 203Pb-TCMC-trastuzumab was the only indicator that cGMP TCMC-trastuzumab may be sensitive to storage at 37 °C for 3 months.

Preparation and Integrity Examination of Freeze Dried Kit of Trastuzumab-Immunoconjugates and Cold Labeled Immunoconjugates by Applying SDS-Page Electrophoresis

“…The cold labeling was done before starting the process of electrophoresis. The obtained results of migration of formed samples correspond to already published results for other IgG1 monoclonal antibodies (18,28). Under reducing conditions, migration of the trastuzumab provided separation of two bands of fragments with molecular weight of 25 kDa for light chain and 50 kDa for heavy chain, proven with the Wide Range, molecular weight marker.…”

“…Many stability studies of trastuzumab were made using a SDS-PAGE in reducing or non-reducing conditions. Under reducing conditions, trastuzumab was migrated as two bands a ∼50 kDa and ∼25 kDa (Mr of heavy and light chain), while under non-reducing conditions only one band at ~150 kDa was observed (Mr of whole antibody) (18,28,29). Our group has already used the applied method of reducing electrophoresis for integrity examinations of rituximab after conjugation and lyophilization (17,19).…”

An Appendix of Radionuclides Used in Targeted Alpha Therapy

“…A derivative of DOTA with carbamoyl methyl arms on the cyclen ring (TCMC) instead of carboxylate arms have been studied in preclinical and clinical settings. A few targeting agents have been conjugated to TCMC to target different cancers and to be administrated through intravenous or intraperitoneal injection [9,[32][33][34].…”

Biokinetic Modeling and Dosimetry for Optimizing Intraperitoneal Radioimmunotherapy of Ovarian Cancer Microtumors

“…Good results would also be expected for 212 Pb-mAb but only if all, instead of the reported 65% (23), of the radionuclide daughters decay where the parent 212 Pb decay. In addition, higher activity concentrations and specific activities would be needed than those reported (24). Use of short-lived a-emitters such as 213 Bi requires high activity concentrations to eradicate microtumors.…”