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.

Tuning monoclonal antibody galactosylation using Raman spectroscopy-controlled lactic acid feeding.

A key aspect of large-scale production of biotherapeutics is a well-designed and consistently-executed upstream cell culture process. Process analytical technology tools provide enhanced monitoring and control capabilities to support consistent process execution, and also have potential to aid in maintenance of product quality at desired levels. One such tool, Raman spectroscopy, has matured as a useful technique to achieve real-time monitoring and control of key cell culture process attributes. We developed a Raman spectroscopy-based nutrient control strategy to enable dual control of lactate and glucose levels for a fed-batch CHO cell culture process for monoclonal antibody (mAb) production. To achieve this, partial least squares-based chemometric models for real-time prediction of glucose and lactate concentrations were developed and deployed in feedback control loops. In particular, feeding of lactic acid post-metabolic shift was investigated based on previous work that has shown the impact of lactate levels on ammonium as well as mAb product quality. Three feeding strategies were assessed for impact on cell metabolism, productivity, and product quality: bolus-fed glucose, glucose control at 4 g/L, or simultaneous glucose control at 4 g/L and lactate control at 2 g/L. The third feeding strategy resulted in a significant reduction in ammonium levels (68%) while increasing mAb galactosylation levels by approximately 50%. This work demonstrated that when deployed in a cell culture process, Raman spectroscopy is an effective technique for simultaneous control of multiple nutrient feeds, and that lactic acid feeding can have a positive impact on both cell metabolism and mAb product quality.

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.

Identifying a robust design space for glycosylation during monoclonal antibody production.

Glycan distribution has been identified as a "critical quality attribute" for many biopharmaceutical products, including monoclonal antibodies. Consequently, determining quantitatively how process variables affect glycan distribution is important during process development to control antibody glycosylation. In this work, we assess the effect of six bioreactor process variables on the glycan distribution of an IgG1 produced in CHO cells. Our analysis established that glucose and glutamine media concentration, temperature, pH, agitation rate, and dissolved oxygen (DO) had small but significant effects on the relative percentage of various glycans. In addition, we assessed glycosylation enzyme transcript levels and intracellular sugar nucleotide concentrations within the CHO cells to provide a biological explanation for the observed effects on glycan distributions. From these results we identified a robust operating region, or design space, in which the IgG1 could be produced with a consistent glycan distribution. Since our results indicate that perturbations to bioreactor process variables will cause only small (even if significant) changes to the relative percentage of various glycans (<±1.5%)-changes that are too small to affect the bioactivity and efficacy of this IgG1 significantly-it follows that the glycan distribution obtained will be consistent even with relatively large variations in bioreactor process variables. However, for therapeutic proteins where bioactivity and efficacy are affected by small changes to the relative percentage of glycans, the same analysis would identify the manipulated variables capable of changing glycan distribution, and hence can be used to implement a glycosylation control strategy. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1149-1162, 2016.

[Chromosome alterations in workers exposed to ionizing radiation]. / Alteraciones cromosómicas en trabajadores expuestos a radiaciones ionizantes.

With the purpose of determining and characterizing chromosomal alterations and their relation to the radiation dose, time of employment and weekly exposure time, a transversal cut-descriptive study was performed on 18 workers, exposed to ionizing radiation, from a specialized company in the Venezuelan oil industry. These workers, male and females, constituted all the population studied, aged between 32 and 59 years, with at least one year on the job. A random sample of a non-exposed group of workers was used as a control. An occupational medical report was applied and personal dosimetry, environmental monitoring and a chromosomal analysis using two chromosomic culture techniques, were performed. The results show, in the exposed groups, an age average of 46.10 +/- 7.69 years, an average 17.5 +/- 5.00 years of employment and a weekly exposure of 4.30 +/- 1.33 hours. In the exposed population, 444 chromosomal abnormalities were evidenced in 700 metaphases studied; these abnormalities consisted of 66.6% single fragilities, 22% of combined fragilities, with chromosomic ruptures, deletions and poliploids, and 11% presented a normal kariotype. The control group presented chromosomic alterations as single fragilities in 55% of the cases. Radiologists presented 88.8% of chromosomic alterations, with below permissible doses detected, and 11.2% of them with exceed doses, presented the greatest number of fragilities and multiple chromosomic ruptures. The radiologists with weekly exposures of 8 hours presented the highest number of chromosomic alterations. 88% of radiologists with chromosomal abnormalities had more than 10 years of exposure. In conclusion, chronic exposure to low levels of ionizing radiation can induce chromosomic alterations, depending on the years of employment and the weekly time of exposure.

Alteraciones cromosómicas en trabajadores expuestos a radiaciones ionizantes / Chromosome alterations in workers exposed to ionizing radiation

Con el objeto de determinar y caracterizar las alteraciones cromosómicas, y el tiempo de exposición semanal a la radiación ionizante, se realizó un estudio descriptivo, de corte transversal, en 18 trabajadores de ambos sexos, que constituyeron la población estudio, con edades entre 32 y 59 años, con un año mínimo de exposición en una empresa especializada en el negocio petrolero venezolano. El grupo control seleccionado al azar simple, no estuvo expuesto a radiación ionizante. Se practicó una historia médico ocupacional, análisis cromosómico utilizando dos técnicas de cultivo cromosómico, dosimetría personal y monitoreo ambiental al grupo expuesto a radiación ionizante. Los resultados mostraron una edad promedio de 46,10 ± 7,69 años en el grupo expuesto, con antigüedad en el puesto de trabajo de 17,5 ± 5,0 años y tiempo de exposición semanal de 4,30 ± 1,33 horas. Se evidenciaron 444 alteraciones cromosómicas en 700 metafases estudiadas en la población expuesta, representadas por fragilidades simples 66,6 por ciento fragilidades combinadas, con rupturas cromosómicas, delecciones y poliploidías en 22,2 por ciento, el 11,1 por ciento presentó cariotipo normal. El grupo control presentó alteraciones cromosómicas tipo fragilidades simples en el 55,5 por ciento. En los radiólogos se observó el 88,8 por ciento de alteraciones cromosómicas, con dosis detectada por debajo de lo permisible, y el 11,2 de ellos, con dosis excedidas, presentó el mayor número de fragilidades y rupturas cromosómicas múltiples. Los radiólogos con exposición semanal de 8 horas mostraron el mayor número de rupturas cromosómicas. Ochenta y ocho por ciento de los radiólogos con alteraciones cromosómicas tenían antigüedades mayor de 10 años de exposición. En conclusión, la exposición crónica a bajas dosis de radiación ionizante puede inducir alteraciones cromosómicas, dependiendo de la antigüedad en la ocupación y la exposición semanal