A Fully Integrated Online Platform For Real Time Monitoring Of Multiple Product Quality Attributes In Biopharmaceutical Processes For Monoclonal Antibody Therapeutics.

In response to FDA's call for Quality by Design (QbD) in biopharmaceutical product development, the biopharmaceutical industry has been developing highly sensitive and specific technologies in the monitoring and controlling of product quality attributes for bioprocesses. We previously published the successful application of an off-line multi-attribute method (MAM) to monitor more than 20 critical quality attributes (CQA) with superior sensitivity for the upstream process. To further remove the hurdles of laborious process sampling and sample preparation associated with the offline method, we present here a fully integrated MAM based online platform for automated real time online process monitoring. This integrated system includes Modular Automated Sampling Technology (MAST) based aseptic sampling, multi-function Sequential Injection Analysis (SIA) sample preparation, UHPLC separation and high-resolution mass spectrometry (HRMS) analysis. Continuous automated daily monitoring of a 17-day cell culture process was successfully demonstrated for a model monoclonal antibody (mAb) molecule with similar specificity and sensitivity as we reported earlier. To the best of our knowledge, this is the first report of an end-to-end automated online MAM system, which would allow the MAM to be applied to routine bioprocess monitoring, potentially replacing multiple conventional low resolution and low sensitivity off-line methods. The online HPLC or HPLC/MS platform could be easily adapted to support other processing steps such as downstream purification with minimal software re-configuration.

Rapid and comprehensive monoclonal antibody Characterization using microfluidic CE-MS.

The identification and control of monoclonal antibody (mAb) critical quality attributes (CQAs) is a key component of quality by design (QbD). In this work, rapid peptide mapping and native intact charge variants analysis have been developed to comprehensively characterize and monitor mAb CQAs using a microfluidic capillary electrophoresis - mass spectrometry (CE-MS) platform. The ultrafast peptide mapping simultaneously analyzed multiple CQAs, including protein primary structure, oxidation, deamidation, succinimide, C-terminal lysine (Lys) clipping, N-terminal cyclization, and glycosylation. The microfluidic CE-MS based peptide mapping acquired results comparable to conventional but lengthy liquid chromatography - MS (LC-MS) based approach. The native intact analysis resolved mAb charge variants with a comparable resolution as commonly achieved using capillary isoelectric focusing (cIEF). Charge variants' identities were assigned based on characteristic mass shifts, knowledge learned from peptide mapping, and changes in electrophoretic mobility. Major mAb glycoforms of each charge variants were resolved and identified in the deconvoluted mass spectra. Furthermore, a model simulation was performed to reconstruct intact deconvoluted mass spectra using peptide mapping results. The reconstructed and experimentally determined intact deconvoluted mass spectra were highly correlated, suggesting that our data collected at the peptide level and intact level were consistent and highly comparable. Overall, the microfluidic CE-MS based peptide mapping and native intact charge variants analysis are high-throughput methods that have great potential to support biopharmaceutical development.

Simultaneous Monitoring and Comparison of Multiple Product Quality Attributes for Cell Culture Processes at Different Scales Using a LC/MS/MS Based Multi-Attribute Method.

In response to the FDA's call for applying Quality by Design (QbD) to the manufacturing process, the biopharmaceutical industry has invested extensively into the monitoring and controlling of product quality attributes for bioprocesses. To assure the safety and efficacy of the drug product, defining critical quality attributes (CQA) and understanding their correlation with critical process parameters (CPP) becomes vitally important. In this work, a liquid chromatography-mass spectrometry based multi-attribute method (MAM) has been applied to the monitoring and trending of multiple CQAs of a monoclonal antibody product. To the best of our knowledge, this is the first demonstration of applying MAM to both a 3-liter development mini-bioreactor (3 L bioreactor) and a 2000-liter GMP single use bioreactor (2000L SUB). MAM was proven not only to be a great analytical tool for monitoring product quality attributes throughout the time course of the cell culture process, it could also provide critical product quality information in order to understand any potential process performance differences during scale-up and/or technology transfer. The successful monitoring and trending of the multiple CQAs throughout the 17-day cell culture process lays a solid foundation for possible real time in-process control and release of biotherapeutics using MAM in the future.

Effects of particle size, food, and capsule shell composition on the oral bioavailability of dabrafenib, a BRAF inhibitor, in patients with BRAF mutation-positive tumors.

Dabrafenib is a small-molecule inhibitor of BRAF kinase activity that is currently being developed for the treatment of BRAF V600 mutation-positive melanoma. This clinical, open-label, two-cohort (n = 14 per cohort), randomized study was designed to evaluate the effect of drug substance particle size, and food on the plasma pharmacokinetics of a single oral dose of dabrafenib in patients with BRAF V600 mutation-positive solid tumors. In addition, an exploratory cross-cohort comparison of the relative bioavailability of single-dose dabrafenib administered in gelatin and hydroxypropyl methylcellulose (HPMC) capsules was performed. Higher bioavailability was noted with nonmicronized drug substance (larger particle size), under fasting conditions, and with HPMC capsules. Initial dissolution results at pH 1.2 showed higher dissolution of gelatin relative to HPMC capsules inconsistent with clinical data. Subsequent in vitro dissolution studies were conducted in fasted-state simulated gastric fluid over a 24-h period and showed that HPMC capsules reached a higher percentage of dabrafenib dissolved than gelatin capsules. The presence of HPMC is believed to inhibit precipitation of dabrafenib as the freebase, thereby maintaining a supersaturated solution over an extended period of time. Dabrafenib has been administered in pivotal clinical studies on an empty stomach using micronized drug substance in HPMC capsules.