Establishment and validation of a microfluidic capillary gel electrophoresis platform method for purity analysis of therapeutic monoclonal antibodies.

Capillary and microfluidic chip electrophoresis technologies are heavily utilized for development, characterization, release, and stability testing of biopharmaceuticals. Within the biopharmaceutical industry, CE-SDS and M-CGE are commonly used for purity determination by separation and quantitation of size-based variants. M-CGE is used primarily as an R&D tool for product and process development, while cGMP release and stability testing applications are commonly reserved for CE-SDS. This paper describes the establishment of an M-CGE platform method to be used for R&D and cGMP applications, including release and stability testing, for monoclonal antibodies. The M-CGE platform method enables testing for product development support and cGMP release and stability using the same method, and utilization of one CE technology for the entire lifecycle of a biopharmaceutical product. Critical method parameters were identified, and the analytical design space of those critical parameters was defined using design of experiments (DOE) studies. Once defined through DOE studies, the method design space was validated according to ICH Q2 (R1) guidelines. Additional molecules of the same validated class were verified for use in the method by experimental confirmation of accuracy, specificity, and stability indicating capabilities. The platform method model facilitates rapid utilization of the method in development and GMP testing environments, and eliminates the need for individual validations for assets of the same class entering early stage development.

An analysis of neurosurgical practice patterns and outcomes for serious to critical traumatic brain injuries in a mature trauma state.

BACKGROUND: We sought to characterize trends in neurosurgical practice patterns and outcomes for serious to critical traumatic brain injuries from 2003 to 2013 in the mature trauma state of Pennsylvania. METHODS: All 2003 to 2013 admissions to Pennsylvania's 30 accredited Level I to II trauma centers with serious to critical traumatic brain injuries (head Abbreviated Injury Scale [AIS] score ≥ 3, Glasgow Coma Scale [GCS] score < 13) were extracted from the state registry. Adjusted temporal trend tests controlling for demographic and injury severity covariates assessed the impact of admission year on intervention rates (craniotomy, craniectomy, and intracranial pressure monitor/ventriculostomy [ICP]) and outcome measures for the total population as well as serious (head AIS score ≥ 3; GCS score, 9-12) and critical (head AIS score ≥ 3, GCS score ≤ 8) subgroups. RESULTS: A total of 22,229 patients met inclusion criteria. Admission year was significantly associated with an adjusted increase in craniectomy (adjusted odds ratio [AOR], 1.12 [1.09-1.14]; p < 0.001) and ICP rates (AOR, 1.03 [1.02-1.04]; p < 0.001) and a decrease in craniotomy rate (AOR, 0.96 [0.95-0.97]; p < 0.001). No significant trends in adjusted mortality were found for the total study population (AOR, 1.01 [1.00-1.02]; p = 0.150); however, a significant reduction was found for the serious subgroup (AOR, 0.95 [0.92-0.98]; p = 0.002), and a significant increase was found for the critical subgroup (AOR, 1.02 [1.01-1.03]; p = 0.004). CONCLUSION: Total study population trends showed a reduction in rates of craniotomy and increase in craniectomy and ICP rates without any change in outcome. Despite significant adaptations in neurosurgical practice patterns from 2003 to 2013, only patients with serious head injuries are experiencing improved survival. LEVEL OF EVIDENCE: Prognostic and epidemiologic study, level III; therapeutic study, level IV.

Suppressing posttranslational gluconoylation of heterologous proteins by metabolic engineering of Escherichia coli.

Minimization of chemical modifications during the production of proteins for pharmaceutical and medical applications is of fundamental and practical importance. The gluconoylation of heterologously expressed protein which is observed in Escherichia coli BL21(DE3) constitutes one such undesired posttranslational modification. We postulated that formation of gluconoylated/phosphogluconoylated products of heterologous proteins is caused by the accumulation of 6-phosphogluconolactone due to the absence of phosphogluconolactonase (PGL) in the pentose phosphate pathway. The results obtained demonstrate that overexpression of a heterologous PGL in BL21(DE3) suppresses the formation of the gluconoylated adducts in the therapeutic proteins studied. When this E. coli strain was grown in high-cell-density fed-batch cultures with an extra copy of the pgl gene, we found that the biomass yield and specific productivity of a heterologous 18-kDa protein increased simultaneously by 50 and 60%, respectively. The higher level of PGL expression allowed E. coli strain BL21(DE3) to satisfy the extra demand for precursors, as well as the energy requirements, in order to replicate plasmid DNA and express heterologous genes, as metabolic flux analysis showed by the higher precursor and NADPH fluxes through the oxidative branch of the pentose phosphate shunt. This work shows that E. coli strain BL21(DE3) can be used as a host to produce three different proteins, a heterodimer of liver X receptors, elongin C, and an 18-kDa protein. This is the first report describing a novel and general strategy for suppressing this nonenzymatic modification by metabolic pathway engineering.

Quantitation of underivatized free amino acids in mammalian cell culture media using matrix assisted laser desorption ionization time-of-flight mass spectrometry.

In this investigation, a quantitative matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) method was developed for the analysis of underivatized free amino acids in mammalian cell culture media. Calibration curves were developed for 12 amino acids over the linear range of 1-100 microM with coefficients of determination ranging from r2 = 0.9220 to r2 = 0.9973. An aerospray method was utilized for the sample deposition method, and the matrix, alpha-cyano-4-hydroxycinnamic acid, served as the internal standard. This assay was used to analyze bioreactor samples from five time points in the process. Concentrations determined through interpolation of the calibration curves were comparable to those obtained via reversed-phase HPLC based analysis with an average percent difference of 19.71%. Repeatability and intermediate precision studies were also performed, and the relative standard deviations ranged from 0.5943 to 21.41 and 3.157 to 18.97, respectively.