Process development and characterization for integrated continuous bioprocesses-Highlights from N-mAb.

The N-mAb case study was produced by the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) to support teaching and learning for both industry and to accelerate adoption of advanced manufacturing process technologies such as integrated continuous bioprocesses (ICB) for mAbs. Similar to the A-mAb case study, N-mAb presents the evolution of an integrated control strategy, from early clinical through process validation and commercial manufacturing with a focus on elements that are unique to integrated continuous bioprocesses. This publication presents a summary of the process design and characterization chapters to allow a greater focus on the unique elements relevant to that phase of development.

Glucose monitoring and adaptive feeding of mammalian cell culture in the presence of strong autofluorescence by near infrared Raman spectroscopy.

Raman spectroscopy offers an attractive platform for real-time monitoring and control of metabolites and feeds in cell culture processes, including mammalian cell culture for biopharmaceutical production. However, specific cell culture processes may generate substantial concentrations of chemical species and byproducts with high levels of autofluorescence when excited with the standard 785 nm wavelength. Shifting excitation further toward the near-infrared allows reduction or elimination of process autofluorescence. We demonstrate such a reduction in a highly autofluorescent mammalian cell culture process. Using the Kaiser RXN2-1000 platform, which utilizes excitation at 993 nm, we developed multivariate glucose models in a cell culture process which was previously impossible using 785 nm excitation. Additionally, the glucose level in the production bioreactor was controlled entirely by Raman adaptive feeding, allowing for maintenance of glucose levels at an arbitrary set point for the duration of the culture. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1574-1580, 2018.

Closed loop control of lactate concentration in mammalian cell culture by Raman spectroscopy leads to improved cell density, viability, and biopharmaceutical protein production.

Accumulation of lactate in mammalian cell culture often negatively impacts culture performance, impeding production of therapeutic proteins. Many efforts have been made to limit the accumulation of lactate in cell culture. Here, we describe a closed loop control scheme based on online spectroscopic measurements of glucose and lactate concentrations. A Raman spectroscopy probe was used to monitor a fed-batch mammalian cell culture and predict glucose and lactate concentrations via multivariate calibration using partial least squares regression (PLS). The PLS models had a root mean squared error of prediction (RMSEP) of 0.27 g/L for glucose and 0.20 g/L for lactate. All glucose feeding was controlled by the Raman PLS model predictions. Glucose was automatically fed when lactate levels were beneath a setpoint (either 4.0 or 2.5 g/L) and glucose was below its own setpoint (0.5 g/L). This control scheme was successful in maintaining lactate levels at an arbitrary setpoint throughout the culture, as compared to the eventual accumulate of lactate to 8.0 g/L in the historical process. Automated control of lactate by restricted glucose feeding led to improvements in culture duration, viability, productivity, and robustness. Culture duration was extended from 11 to 13 days, and harvest titer increased 85% over the historical process. Biotechnol. Bioeng. 2016;113: 2416-2424. © 2016 Wiley Periodicals, Inc.

Mechanism investigation for poloxamer 188 raw material variation in cell culture.

Variability in poloxamer 188 (P188) raw material, which is routinely used in cell culture media to protect cells from hydrodynamic forces, plays an important role in the process performance. Even though tremendous efforts have been spent to understand the mechanism of poloxamer's protection, the root cause for lot-to-lot variation was not clear. A recent study reported that the low performance was not due to toxicity but inefficiency to protect cells (Peng et al., Biotechnol Prog. 2014;30:1411-1418). In this study, it was demonstrated for the first time that the addition of other surfactants even at a very low level can interfere with P188 resulting in a loss of efficiency. It was also found that the performance of P188 lots correlated well with its foam stability. Foam generated from low performing lots in baffled shaker flask lasts longer, which suggests that the components in the foam layers are different. The spiking of foam generated from a low performing lot into the media containing a high performance lot resulted in cell damage and low growth. Analytical studies using size exclusion chromatography (SEC) identified differences in high molecular weight (HMW) species present in the P188 lots. These differences are much clearer when comparing the HMW region of the SEC chromatogram of foam vs. bulk liquid samples. This study shows that low performing lots have enriched HMW species in foam samples due to high hydrophobicity, which can be potentially used as a screening assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:767-775, 2016.

Quick generation of Raman spectroscopy based in-process glucose control to influence biopharmaceutical protein product quality during mammalian cell culture.

Mitigating risks to biotherapeutic protein production processes and products has driven the development of targeted process analytical technology (PAT); however implementing PAT during development without significantly increasing program timelines can be difficult. The development of a monoclonal antibody expressed in a Chinese hamster ovary (CHO) cell line via fed-batch processing presented an opportunity to demonstrate capabilities of altering percent glycated protein product. Glycation is caused by pseudo-first order, non-enzymatic reaction of a reducing sugar with an amino group. Glucose is the highest concentration reducing sugar in the chemically defined media (CDM), thus a strategy controlling glucose in the production bioreactor was developed utilizing Raman spectroscopy for feedback control. Raman regions for glucose were determined by spiking studies in water and CDM. Calibration spectra were collected during 8 bench scale batches designed to capture a wide glucose concentration space. Finally, a PLS model capable of translating Raman spectra to glucose concentration was built using the calibration spectra and spiking study regions. Bolus feeding in mammalian cell culture results in wide glucose concentration ranges. Here we describe the development of process automation enabling glucose setpoint control. Glucose-free nutrient feed was fed daily, however glucose stock solution was fed as needed according to online Raman measurements. Two feedback control conditions were executed where glucose was controlled at constant low concentration or decreased stepwise throughout. Glycation was reduced from ∼9% to 4% using a low target concentration but was not reduced in the stepwise condition as compared to the historical bolus glucose feeding regimen.

Cross-scale predictive modeling of CHO cell culture growth and metabolites using Raman spectroscopy and multivariate analysis.

Multi-component, multi-scale Raman spectroscopy modeling results from a monoclonal antibody producing CHO cell culture process including data from two development scales (3 L, 200 L) and a clinical manufacturing scale environment (2,000 L) are presented. Multivariate analysis principles are a critical component to partial least squares (PLS) modeling but can quickly turn into an overly iterative process, thus a simplified protocol is proposed for addressing necessary steps including spectral preprocessing, spectral region selection, and outlier removal to create models exclusively from cell culture process data without the inclusion of spectral data from chemically defined nutrient solutions or targeted component spiking studies. An array of single-scale and combination-scale modeling iterations were generated to evaluate technology capabilities and model scalability. Analysis of prediction errors across models suggests that glucose, lactate, and osmolality are well modeled. Model strength was confirmed via predictive validation and by examining performance similarity across single-scale and combination-scale models. Additionally, accurate predictive models were attained in most cases for viable cell density and total cell density; however, these components exhibited some scale-dependencies that hindered model quality in cross-scale predictions where only development data was used in calibration. Glutamate and ammonium models were also able to achieve accurate predictions in most cases. However, there are differences in the absolute concentration ranges of these components across the datasets of individual bioreactor scales. Thus, glutamate and ammonium PLS models were forced to extrapolate in cases where models were derived from small scale data only but used in cross-scale applications predicting against manufacturing scale batches.

On-chip microelectrode impedance analysis of mammalian cell viability during biomanufacturing.

The characterization of cell viability is a challenging task in applied biotechnology, as no clear definition of cell death exists. Cell death is accompanied with a change in the electrical properties of the membrane as well as the cell interior. Therefore, changes in the physiology of cells can be characterized by monitoring of their dielectric properties. We correlated the dielectric properties of industrially used mammalian cells, sedimented over interdigitated microelectrodes, to the AC signal response across the chip. The voltage waveforms across the electrodes were processed to obtain the circuit impedance, which was used to quantify the changes in cell viability. We observed an initial decrease in impedance, after which it remained nearly constant. The results were compared with data from the dye exclusion viability test, the cell specific oxygen uptake rate, and the online viable cell density data from capacitance probes. The microelectrode technique was found to be sensitive to physiological changes taking place inside the cells before their membrane integrity is compromised. Such accurate determination of the metabolic status during this initial period, which turned out to be less well captured in the dye exclusion tests, may be essential for several biotechnology operations.

Development of small scale cell culture models for screening poloxamer 188 lot-to-lot variation.

Shear protectants such as poloxamer 188 play a critical role in protecting cells during cell culture bioprocessing. Lot-to-lot variation of poloxamer 188 was experienced during a routine technology transfer across sites of similar scale and equipment. Cell culture medium containing a specific poloxamer 188 lot resulted in an unusual drop in cell growth, viability, and titer during manufacturing runs. After switching poloxamer lots, culture performance returned to the expected level. In order to control the quality of poloxamer 188 and thus maintain better consistency in manufacturing, multiple small scale screening models were developed. Initially, a 5L bioreactor model was established to evaluate cell damage by high sparge rates with different poloxamer 188 lots. Subsequently, a more robust, simple, and efficient baffled shake flask model was developed. The baffled shake flask model can be performed in a high throughput manner to investigate the cell damage in a bubbling environment. The main cause of the poor performance was the loss of protection, rather than toxicity. It was also suggested that suspicious lots can be identified using different cell line and media. The screening methods provide easy, yet remarkable models for understanding and controlling cell damage due to raw material lot variation as well as studying the interaction between poloxamer 188 and cells.

Effects of bubble­liquid two-phase turbulent hydrodynamics on cell damage in sparged bioreactor.

According to recent experimental studies on sparged bioreactors, significant cell damage may occur at the gas inlet region near the sparger. Although shear stress was proposed to be one of the potential causes for cell damage, detailed hydrodynamic studies at the gas inlet region of gas­liquid bioreactors have not been performed to date. In this work, a second-order moment (SOM) bubble­liquid two-phase turbulent model based on the two-fluid continuum approach is used to investigate the gas­liquid hydrodynamics in the bubble column reactor and their potential impacts on cell viability, especially at the gas inlet region. By establishing fluctuation velocity and bubble­liquid two-phase fluctuation velocities correlation transport equations, the anisotropy of two-phase stresses and the bubble­ liquid interactions are fully considered. Simulation results from the SOM model indicate that shear and normal stresses, turbulent energy dissipation rate, and the turbulent kinetic energy are generally smaller at the gas inlet region when compared with those in the fully developed region. In comparison, a newly proposed correlation expression, stress-induced turbulent energy production (STEP), is found to correlate well with the unusually high cell death rate at the gas inlet region. Therefore, STEP, which represents turbulent energy transfer to a controlled volume induced by a combination of shear and normal stresses, has the potential to provide better explanation for increased cell death at the sparger region.

Performance of high intensity fed-batch mammalian cell cultures in disposable bioreactor systems.

The adoption of disposable bioreactor technology as an alternate to traditional nondisposable technology is gaining momentum in the biotechnology industry. Evaluation of current disposable bioreactors systems to sustain high intensity fed-batch mammalian cell culture processes needs to be explored. In this study, an assessment was performed comparing single-use bioreactors (SUBs) systems of 50-, 250-, and 1,000-L operating scales with traditional stainless steel (SS) and glass vessels using four distinct mammalian cell culture processes. This comparison focuses on expansion and production stage performance. The SUB performance was evaluated based on three main areas: operability, process scalability, and process performance. The process performance and operability aspects were assessed over time and product quality performance was compared at the day of harvest. Expansion stage results showed disposable bioreactors mirror traditional bioreactors in terms of cellular growth and metabolism. Set-up and disposal times were dramatically reduced using the SUB systems when compared with traditional systems. Production stage runs for both Chinese hamster ovary and NS0 cell lines in the SUB system were able to model SS bioreactors runs at 100-, 200-, 2,000-, and 15,000-L scales. A single 1,000-L SUB run applying a high intensity fed-batch process was able to generate 7.5 kg of antibody with comparable product quality.