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4 pages, 489 KiB

Open AccessEditorial

Measurement Technologies for Upstream and Downstream Bioprocessing

by Carl-Fredrik Mandenius

Processes 2021, 9(1), 143; https://doi.org/10.3390/pr9010143 - 13 Jan 2021

Cited by 2 | Viewed by 5088

Abstract

This special issue is devoted to new developments in measurement technologies for upstream and downstream bioprocessing [...] Full article

(This article belongs to the Special Issue Measurement Technologies for up- and Downstream Bioprocessing)

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Research

Jump to: Editorial

18 pages, 1538 KiB

Open AccessFeature PaperArticle

Dielectric Spectroscopy to Improve the Production of rAAV Used in Gene Therapy

by Daniel A. M. Pais, Chris Brown, Anastasia Neuman, Krishanu Mathur, Inês A. Isidro, Paula M. Alves and Peter G. Slade

Processes 2020, 8(11), 1456; https://doi.org/10.3390/pr8111456 - 13 Nov 2020

Cited by 8 | Viewed by 4466

Abstract

The insect cell-baculovirus expression vector system is an established method for large scale recombinant adeno-associated virus (rAAV) production, largely due to its scalability and high volumetric productivities. During rAAV production it is critical to monitor process parameters such as Spodoptera frugiperda (Sf9) cell [...] Read more.

The insect cell-baculovirus expression vector system is an established method for large scale recombinant adeno-associated virus (rAAV) production, largely due to its scalability and high volumetric productivities. During rAAV production it is critical to monitor process parameters such as Spodoptera frugiperda (Sf9) cell concentration, infection timing, and cell harvest viabilities since they can have a significant influence on rAAV productivity and product quality. Herein we developed the use of dielectric spectroscopy as a process analytical technology (PAT) tool used to continuously monitor the production of rAAV in 2 L stirred tank bioreactors, achieving enhanced control over the production process. This study resulted in improved manufacturing robustness through continuous monitoring of cell culture parameters, eliminating sampling needs, increasing the accuracy of infection timing, and reliably estimating the time of harvest. To increase the accuracy of baculovirus infection timing, the cell growth/permittivity model was coupled to a feedback loop with real-time monitoring. This system was able to predict baculovirus infection timing up to 24 h in advance for greatly improved accuracy of infection and ensuring consistent high rAAV productivities. Furthermore, predictive models were developed based on the dielectric measurements of the culture. These multiple linear regression-based models resulted in correlation coefficients (Q²) of 0.89 for viable cell concentration, 0.97 for viability, and 0.92 for cell diameter. Finally, models were developed to predict rAAV titer providing the capability to distinguish in real time between high and low titer production batches. Full article

(This article belongs to the Special Issue Measurement Technologies for up- and Downstream Bioprocessing)

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12 pages, 4140 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Real-Time Nanoplasmonic Sensor for IgG Monitoring in Bioproduction

by Thuy Tran, Olof Eskilson, Florian Mayer, Robert Gustavsson, Robert Selegård, Ingemar Lundström, Carl-Fredrik Mandenius, Erik Martinsson and Daniel Aili

Processes 2020, 8(10), 1302; https://doi.org/10.3390/pr8101302 - 16 Oct 2020

Cited by 16 | Viewed by 4164

Abstract

Real-time monitoring of product titers during process development and production of biotherapeutics facilitate implementation of quality-by-design principles and enable rapid bioprocess decision and optimization of the production process. Conventional analytical methods are generally performed offline/at-line and, therefore, are not capable of generating real-time [...] Read more.

Real-time monitoring of product titers during process development and production of biotherapeutics facilitate implementation of quality-by-design principles and enable rapid bioprocess decision and optimization of the production process. Conventional analytical methods are generally performed offline/at-line and, therefore, are not capable of generating real-time data. In this study, a novel fiber optical nanoplasmonic sensor technology was explored for rapid IgG titer measurements. The sensor combines localized surface plasmon resonance transduction and robust single use Protein A-modified sensor chips, housed in a flexible flow cell, for specific IgG detection. The sensor requires small sample volumes (1–150 µL) and shows a reproducibility and sensitivity comparable to Protein G high performance liquid chromatography-ultraviolet (HPLC-UV). The dynamic range of the sensor system can be tuned by varying the sample volume, which enables quantification of IgG samples ranging from 0.0015 to 10 mg/mL, without need for sample dilution. The sensor shows limited interference from the sample matrix and negligible unspecific protein binding. IgG titers can be rapidly determined in samples from filtered unpurified Chinese hamster ovary (CHO) cell cultures and show good correlation with enzyme-linked immunosorbent assay (ELISA). Full article

(This article belongs to the Special Issue Measurement Technologies for up- and Downstream Bioprocessing)

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31 pages, 4801 KiB

Open AccessFeature PaperArticle

High-Throughput Raman Spectroscopy Combined with Innovate Data Analysis Workflow to Enhance Biopharmaceutical Process Development

by Stephen Goldrick, Alexandra Umprecht, Alison Tang, Roman Zakrzewski, Matthew Cheeks, Richard Turner, Aled Charles, Karolina Les, Martyn Hulley, Chris Spencer and Suzanne S. Farid

Processes 2020, 8(9), 1179; https://doi.org/10.3390/pr8091179 - 17 Sep 2020

Cited by 24 | Viewed by 8634

Abstract

Raman spectroscopy has the potential to revolutionise many aspects of biopharmaceutical process development. The widespread adoption of this promising technology has been hindered by the high cost associated with individual probes and the challenge of measuring low sample volumes. To address these issues, [...] Read more.

Raman spectroscopy has the potential to revolutionise many aspects of biopharmaceutical process development. The widespread adoption of this promising technology has been hindered by the high cost associated with individual probes and the challenge of measuring low sample volumes. To address these issues, this paper investigates the potential of an emerging new high-throughput (HT) Raman spectroscopy microscope combined with a novel data analysis workflow to replace off-line analytics for upstream and downstream operations. On the upstream front, the case study involved the at-line monitoring of an HT micro-bioreactor system cultivating two mammalian cell cultures expressing two different therapeutic proteins. The spectra generated were analysed using a partial least squares (PLS) model. This enabled the successful prediction of the glucose, lactate, antibody, and viable cell density concentrations directly from the Raman spectra without reliance on multiple off-line analytical devices and using only a single low-volume sample (50–300 μL). However, upon the subsequent investigation of these models, only the glucose and lactate models appeared to be robust based upon their model coefficients containing the expected Raman vibrational signatures. On the downstream front, the HT Raman device was incorporated into the development of a cation exchange chromatography step for an Fc-fusion protein to compare different elution conditions. PLS models were derived from the spectra and were found to predict accurately monomer purity and concentration. The low molecular weight (LMW) and high molecular weight (HMW) species concentrations were found to be too low to be predicted accurately by the Raman device. However, the method enabled the classification of samples based on protein concentration and monomer purity, allowing a prioritisation and reduction in samples analysed using A280 UV absorbance and high-performance liquid chromatography (HPLC). The flexibility and highly configurable nature of this HT Raman spectroscopy microscope makes it an ideal tool for bioprocess research and development, and is a cost-effective solution based on its ability to support a large range of unit operations in both upstream and downstream process operations. Full article

(This article belongs to the Special Issue Measurement Technologies for up- and Downstream Bioprocessing)

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16 pages, 4330 KiB

Open AccessFeature PaperArticle

A Reliable Automated Sampling System for On-Line and Real-Time Monitoring of CHO Cultures

by Alexandra Hofer, Paul Kroll, Matthias Barmettler and Christoph Herwig

Processes 2020, 8(6), 637; https://doi.org/10.3390/pr8060637 - 27 May 2020

Cited by 8 | Viewed by 4420

Abstract

Timely monitoring and control of critical process parameters and product attributes are still the basic tasks in bioprocess development. The current trend of automation and digitization in bioprocess technology targets an improvement of these tasks by reducing human error and increasing through-put. The [...] Read more.

Timely monitoring and control of critical process parameters and product attributes are still the basic tasks in bioprocess development. The current trend of automation and digitization in bioprocess technology targets an improvement of these tasks by reducing human error and increasing through-put. The gaps in such automation procedures are still the sampling procedure, sample preparation, sample transfer to analyzers, and the alignment of process and sample data. In this study, an automated sampling system and the respective data management software were evaluated for system performance; applicability with HPLC for measurement of vitamins, product and amino acids; and applicability with a biochemical analyzer. The focus was especially directed towards the adaptation and assessment of an appropriate amino acid method, as these substances are critical in cell culture processes. Application of automated sampling in a CHO fed-batch revealed its potential with regard to data evaluation. The higher sampling frequency compared to manual sampling increases the generated information content, which allows easier interpretation of the metabolism, extraction of e.g., k_s values, application of smoothing algorithms, and more accurate detection of process events. A comparison with sensor technology shows the advantages and disadvantages in terms of measurement errors and measurement frequency. Full article

(This article belongs to the Special Issue Measurement Technologies for up- and Downstream Bioprocessing)

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16 pages, 3046 KiB

Open AccessArticle

Novel Carbon Dioxide-Based Method for Accurate Determination of pH and pCO₂ in Mammalian Cell Culture Processes

by Christian Klinger, Verena Trinkaus and Tobias Wallocha

Processes 2020, 8(5), 520; https://doi.org/10.3390/pr8050520 - 28 Apr 2020

Cited by 6 | Viewed by 6327

Abstract

In mammalian cell culture, especially in pharmaceutical manufacturing, pH is a critical process parameter that has to be controlled as accurately as possible. Not only does pH directly affect cell culture performance, ensuring a comparable pH is also crucial for scaling and transfer [...] Read more.

In mammalian cell culture, especially in pharmaceutical manufacturing, pH is a critical process parameter that has to be controlled as accurately as possible. Not only does pH directly affect cell culture performance, ensuring a comparable pH is also crucial for scaling and transfer of processes. A sample-based offline pH measurement is commonly used to ensure correct bioreactor pH probe signals after sterilization and as a detection measure for drifts of probe signals. However, the sample-based pH offline measurement does not necessarily deliver required accuracy. Offsets between bioreactor pH and sample pH heavily depend on equipment, local procedures and the offline measurement method that is used. This article adequately describes a novel, non-invasive method to determine pH and pCO₂ in sterile bioreactors without the need to sample and measure offline. This method utilizes the chemical correlation between carbon dioxide in the gas phase, dissolved carbon dioxide, bicarbonate and dependent proton concentrations that directly affect the pH in carbonate buffered systems. The proposed carbon dioxide-based pH reference method thereby is able to accurately determine the true pH in the bioreactor without the need to sample. The proposed method is independent of scale and bioreactor configuration and does not depend on local procedures that may differ between sites, scales or operators. Applicability of the method for both stainless steel and single use bioreactors is shown. Furthermore, the very same principles are applicable for non-invasive, online pCO₂ monitoring. Full article

(This article belongs to the Special Issue Measurement Technologies for up- and Downstream Bioprocessing)

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16 pages, 2478 KiB

Open AccessArticle

Holographic Imaging of Insect Cell Cultures: Online Non-Invasive Monitoring of Adeno-Associated Virus Production and Cell Concentration

by Daniel A. M. Pais, Paulo R. S. Galrão, Anastasiya Kryzhanska, Jérémie Barbau, Inês A. Isidro and Paula M. Alves

Processes 2020, 8(4), 487; https://doi.org/10.3390/pr8040487 - 22 Apr 2020

Cited by 14 | Viewed by 5323

Abstract

The insect cell-baculovirus vector system has become one of the favorite platforms for the expression of viral vectors for vaccination and gene therapy purposes. As it is a lytic system, it is essential to balance maximum recombinant product expression with harvest time, minimizing [...] Read more.

The insect cell-baculovirus vector system has become one of the favorite platforms for the expression of viral vectors for vaccination and gene therapy purposes. As it is a lytic system, it is essential to balance maximum recombinant product expression with harvest time, minimizing product exposure to detrimental proteases. With this purpose, new bioprocess monitoring solutions are needed to accurately estimate culture progression. Herein, we used online digital holographic microscopy (DHM) to monitor bioreactor cultures of Sf9 insect cells. Batches of baculovirus-infected Sf9 cells producing recombinant adeno-associated virus (AAV) and non-infected cells were used to evaluate DHM prediction capabilities for viable cell concentration, culture viability and AAV titer. Over 30 cell-related optical attributes were quantified using DHM, followed by a forward stepwise regression to select the most significant (p < 0.05) parameters for each variable. We then applied multiple linear regression to obtain models which were able to predict culture variables with root mean squared errors (RMSE) of 7 × 10⁵ cells/mL, 3% for cell viability and 2 × 10³ AAV/cell for 3-fold cross-validation. Overall, this work shows that DHM can be implemented for online monitoring of Sf9 concentration and viability, also permitting to monitor product titer, namely AAV, or culture progression in lytic systems, making it a valuable tool to support the time of harvest decision and for the establishment of controlled feeding strategies. Full article

(This article belongs to the Special Issue Measurement Technologies for up- and Downstream Bioprocessing)

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15 pages, 2839 KiB

Open AccessFeature PaperArticle

A Chemometric Tool to Monitor and Predict Cell Viability in Filamentous Fungi Bioprocesses Using UV Chromatogram Fingerprints

by Philipp Doppler, Lukas Veiter, Oliver Spadiut, Christoph Herwig and Vignesh Rajamanickam

Processes 2020, 8(4), 461; https://doi.org/10.3390/pr8040461 - 14 Apr 2020

Cited by 6 | Viewed by 3399

Abstract

Monitoring process variables in bioprocesses with complex expression systems, such as filamentous fungi, requires a vast number of offline methods or sophisticated inline sensors. In this respect, cell viability is a crucial process variable determining the overall process performance. Thus, fast and precise [...] Read more.

Monitoring process variables in bioprocesses with complex expression systems, such as filamentous fungi, requires a vast number of offline methods or sophisticated inline sensors. In this respect, cell viability is a crucial process variable determining the overall process performance. Thus, fast and precise tools for identification of key process deviations or transitions are needed. However, such reliable monitoring tools are still scarce to date or require sophisticated equipment. In this study, we used the commonly available size exclusion chromatography (SEC) HPLC technique to capture impurity release information in Penicillium chrysogenum bioprocesses. We exploited the impurity release information contained in UV chromatograms as fingerprints for development of principal component analysis (PCA) models to descriptively analyze the process trends. Prediction models using well established approaches, such as partial least squares (PLS), orthogonal PLS (OPLS) and principal component regression (PCR), were made to predict the viability with model accuracies of 90% or higher. Furthermore, we demonstrated the platform applicability of our method by monitoring viability in a Trichoderma reesei process for cellulase production. We are convinced that this method will not only facilitate monitoring viability of complex bioprocesses but could also be used for enhanced process control with hybrid models in the future. Full article

(This article belongs to the Special Issue Measurement Technologies for up- and Downstream Bioprocessing)

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18 pages, 3760 KiB

Open AccessFeature PaperArticle

Single-Use Printed Biosensor for L-Lactate and Its Application in Bioprocess Monitoring

by Lorenz Theuer, Judit Randek, Stefan Junne, Peter Neubauer, Carl-Fredrik Mandenius and Valerio Beni

Processes 2020, 8(3), 321; https://doi.org/10.3390/pr8030321 - 9 Mar 2020

Cited by 8 | Viewed by 4074

Abstract

There is a profound need in bioprocess manufacturing for low-cost single-use sensors that allow timely monitoring of critical product and production attributes. One such opportunity is screen-printed enzyme-based electrochemical sensors, which have the potential to enable low-cost online and/or off-line monitoring of specific [...] Read more.

There is a profound need in bioprocess manufacturing for low-cost single-use sensors that allow timely monitoring of critical product and production attributes. One such opportunity is screen-printed enzyme-based electrochemical sensors, which have the potential to enable low-cost online and/or off-line monitoring of specific parameters in bioprocesses. In this study, such a single-use electrochemical biosensor for lactate monitoring is designed and evaluated. Several aspects of its fabrication and use are addressed, including enzyme immobilization, stability, shelf-life and reproducibility. Applicability of the biosensor to off-line monitoring of bioprocesses was shown by testing in two common industrial bioprocesses in which lactate is a critical quality attribute (Corynebacterium fermentation and mammalian Chinese hamster ovary (CHO) cell cultivation). The specific response to lactate of the screen-printed biosensor was characterized by amperometric measurements. The usability of the sensor at typical industrial culture conditions was favorably evaluated and benchmarked with commonly used standard methods (HPLC and enzymatic kits). The single-use biosensor allowed fast and accurate detection of lactate in prediluted culture media used in industrial practice. The design and fabrication of the biosensor could most likely be adapted to several other critical bioprocess analytes using other specific enzymes. This makes this single-use screen-printed biosensor concept a potentially interesting and versatile tool for further applications in bioprocess monitoring. Full article

(This article belongs to the Special Issue Measurement Technologies for up- and Downstream Bioprocessing)

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