Development of generic raman models for a GS-KOTM CHO platform process.

This paper concerns real-time, multi-rate, nonlinear state and parameter estimation in a pilot-scale biochemical reactor in which cultivation of mouse-mouse hybridoma cells takes place. A multi-rate estimator is designed and implemented to estimate specific growth rate and concentrations of viable cells, total cells, glucose, glutamine, and monoclonal antibodies (MAb) in the reactor. These are estimated from frequent measurement (inferred values) of oxygen uptake rate (OUR) and infrequent and delayed measurements of the concentrations of viable cells, total cells, glucose, glutamine, and MAb. The infrequent measurements are available every 2 to 17 h with a time delay of 0.08 to 2.00 h, and OUR is inferred from dissolved oxygen concentration measurements that are available very 0.17 h. For each of the process variables, its infrequent measurement data and the profile of its estimate are presented to show the performance of the multi-rate estimator.

Effects on Plasma Glucose and Lactate

Background After selection of a recombinant cell line for the production of a therapeutic protein, attempts are often made to increase volumetric productivity. Various techniques have been employed during the production process when trying to increase product concentration. Some aim to increase the time integral of viable cell concentration (IVC; the number of hours viable cells are available to produce the product) by increasing the maximum viable cell concentration and maintaining high viabilities. Techniques include (i) optimization of media and feeds, (ii) optimization of feeding strategies and (iii) genetic manipulation. Others aim to increase specific production rate (QP) by the deliberate inhibition of cell growth (controlled proliferation). Three methods commonly used to control proliferation are use of (i) chemical agents, (ii) hypothermic conditions and (iii) genetic manipulation. In this study, we focused on increasing volumetric productivity by manipulating QP; in particular, by the use of chemical agents. As a cell line has typically already been selected as a manufacturing cell line prior to assessing such methods to increase QP, the likelihood of success is not predictable: resulting in the frequently heard comment that results are ‘cell line specific’. But what if we were to look at these methods with many cell lines, at a much earlier stage of development (before the final cell line has been selected)? It could be that the cell line with the highest product concentration is typically a non-responder. The work described looks to answer the questions: (1) To what extent does the response to such methods vary in a large panel of cell lines producing the same antibody? and (2) Would their use in an earlier stage of cell line development enable the selection of a ‘better’ manufacturing cell line?

The use of spectroscopic sensors for bioprocess monitoring is a powerful tool within the process analytical technology (PAT) initiative of the US Food and Drug Administration. Spectroscopic sensors enable the simultaneous real-time bioprocess monitoring of various critical process parameters including biological, chemical, and physical variables during the entire biotechnological production process. This potential can be realized through the combination of spectroscopic measurements (UV/Vis spectroscopy, IR spectroscopy, fluorescence spectroscopy, and Raman spectroscopy) with multivariate data analysis to obtain relevant process information out of an enormous amount of data. This review summarizes the newest results from science and industry after the establishment of the PAT initiative and gives a critical overview of the most common in-line spectroscopic techniques. Examples are provided of the wide range of possible applications in upstream processing and downstream processing of spectroscopic sensors for real-time monitoring to optimize productivity and ensure product quality in the pharmaceutical industry.

Aim. Optimization of culture conditions of the probiotic strain Lactobacillus acidophilus 55 in the medium with sodium selenite. Methods. Studies of the influence of sodium selenite, inoculum size and cultivation time on the concentration of viable cells of L. acidophilus 55 were performed using a three-level factorial experiment on Box-Behnken (BBD). Results. It has been found that the inoculum size is the factor that has the greatest effect on the concentration of viable cells. The optimum conditions to maximize the concentration of viable cells in the presence of selenium ions were: the content of sodium selenite in the medium of 8.3%, the amount of inoculum 5.3% and 18 hours of cultivation time. Conclusions. When L. acidophilus 55 was cultured at optimized conditions, the highest concentration of viable cells was 2.5×108 CFU/ml.

The steady-state performance of a multistage fermentor with cell filtering through a membrane and recycling of concentrated cells is studied numerically for continuous production of acetic acid. The tanks-in-series model is used to describe the flow behaviour of culture broth in the multistage fermentor. Kinetic expressions and parameter values are taken from the literature. The effects of the total number of stages, the dilution rate, the bleed ratio, the recycle ratio and the feed concentrations as operating conditions on fermentor characteristics such as the concentrations of total and viable cells as well as substrate and product in each stage, the cell viability, the acetic acid productivity and the substrate conversion were examined under the conditions of equal tank volumes and a recycle ratio equal to or greater than one. An increase in the number of stages and decreases in the bleed and recycle ratios and in the feed concentration of product enhance the productivity. The relations between the maximum productivity and the corresponding optimum dilution rate and between the corresponding outlet concentrations of the four components and the optimum dilution rate are presented as figures. Equations for the average cell viability and the critical dilution rate causing cell washout are also obtained. Furthermore, the minimum feed concentration of substrate for its complete consumption is mentioned. Compared to a single tank fermentor, staging and cell recycle increase substrate utilisation and acetic acid productivity. The computed performance of the fermentor configuration proposed in this study is sufficiently promising for the continuous production of acetic acid that it should now be checked experimentally.

The process analytical technology (PAT) initiative is now 10 years old. This has resulted in the development of many tools and software packages dedicated to PAT application on pharmaceutical processes. However, most applications are restricted to small molecule drugs, mainly for the relatively simple process steps like drying or tableting where only a limited number of parameters need to be controlled. A big challenge for PAT still lies in applications for biopharmaceuticals and then especially in the cultivation process step, where the quality of a biopharmaceutical product is largely determined. This review gives an overview of the currently available tools for monitoring and controlling the biopharmaceutical cultivation step and of the main challenges for the most common cell platforms (i.e. Escherichia coli, yeast, and mammalian cells) used in biopharmaceutical manufacturing. The real challenge is to understand how intracellular mechanisms (from synthesis to excretion) influence the quality of biopharmaceuticals and how these mechanisms can be monitored and controlled to yield the desired end product quality. Modern “omics” tools and advanced process analyzers have opened up the way for PAT applications for the biopharmaceutical cultivation process step.

At present time, on-line monitoring of bioprocesses is still one of the most challenging research areas in biochemical engineering. While most industrial fermentors can be controlled by monitoring such extracellular parameters such as dissolved oxygen, pH and temperature, the most important bioprocess parameters, cell concentration and cellular metabolic state as well as substrate and product concentrations cannot be monitored directly on-line. This has limited the control and optimization of bioprocesses.

Process analytical tools for monitoring, understanding, and control of pharmaceutical fluidized bed granulation: A review

Mathematical modeling is at the heart of most current developments in biological system analysis and bioprocess optimization and control. At the industrial scale, this evolution is reflected in process analytical technologies (PAT), digital twins, and Industry 4.0. This book focuses on various aspects of mathematical modeling at the microscopic and macroscopic scales, respectively, and demonstrates the potential of these methodologies to gain insight into the cell metabolism, to support the design of software sensors to reconstruct unmeasurable variables, or to establish model-based optimization of the operating conditions and/or feedback control of the bioprocesses. The range of applications is vast, including biopharmaceuticals, bioenergy, and the environment.

Since the permittivity signal of a cell suspension measured using dielectric spectroscopy at radio frequencies is essentially determined only by viable (intact) cells, it can be used to monitor the concentration of viable cells in a fermentor in which a large proportion of the cells is nonviable. This could be used to select for organisms that are highly resistant to stress, for example from toxic chemicals used in biotransformations. We sought to control the concentration of viable yeast cells in a fermentor by adding small amounts of benzaldehyde, thus imposing a selection regime for cells highly resistant to benzaldehyde. However, after the addition of benzaldehyde, an increase in the permittivity is seen first followed by a decrease, thus making the control of biomass using a standard on-off controller difficult. It is shown that it is possible effectively to control the level of viable biomass in the fermentor in the presence of a large concentration of necromass using a combination of an inverse response compensator and a PID controller.

Propidium monoazide (PMA) is a selective nucleic acid intercalating dye that can be combined with real-time PCR (qPCR) in order to evaluate cell viability in food samples. The aim of the present work was to evaluate PMA-qPCR to detect and quantify viable STEC cells in beef burgers using stx2 as target gene. First, it was determined that 100 µM of PMA could inhibit qPCR signal from non-viable cells and had no influence on the amplification of different concentrations of viable cells. Then, it was shown that PMA efficiently distinguished between different log cfu of viable cells in presence of a high concentration of non-viable cells, both in culture and in beef burger homogenates. Finally, it was determined that PMA could distinguish between viable and non-viable cells within the same log cfu in beef burger homogenates. PMA-qPCR effectively detected and quantified viable STEC cells in culture and in beef burger homogenates. Novelty impact statement The main achievement of this work is that we demonstrate PMA-qPCR could not only detect, but also quantify viable STEC cells targeting stx2 gene, even in the presence of a high concentration of non-viable STEC cells in a complex matrix as beef burgers. This methodology can be used to assess effectiveness of antimicrobial treatments to reduce STEC contamination in meat products more rapidly and with less pathogenic residues than conventional methods.

Process analytical technology (PAT), though a viable field for several decades, is finally com ing of age in the pharmaceuti cal industry. The uS FDA’s PAT initiative of 2004 has led to the rebirth of a field that has proven itself in other industries with the potential of gaining manufacturing efficien cies and better regulation for pharmaceu tical manufacturing processes. When one looks at the role of spectroscopy in PAT, it is clear that there are huge opportunities for increasing process understanding by use of such tools throughout the development of processes and on into manufacturing, where the development knowledge can be applied to provide process verification and control. In this article, examples of how NIR spectroscopy is used in various areas of the pharmaceutical industry will be discussed. however, since PAT has yet to be widely adopted in the pharmaceutical industry, there is still a stage of NIR education and development for such applications, though the science may be well understood. As NIR instrumentation and software development continues to progress, the applications and implementations of NIR in these areas are bound to increase. In the manufacture of active pharmaceuti cal ingredients (APIs), there are many exam ples of spectroscopy deployment and these have been well described.

Continuous beer fermentation by high cell-density culture of bottom brewer's yeast

SummaryRecently, syngas has gained significant interest as renewable and sustainable feedstock, in particular for the biotechnological production of poly([R]‐3‐hydroxybutyrate) (PHB). PHB is a biodegradable, biocompatible polyester produced by some bacteria growing on the principal component of syngas, CO. However, working with syngas is challenging because of the CO toxicity and the explosion danger of H2, another main component of syngas. In addition, the bioprocess control needs specific monitoring tools and analytical methods that differ from standard fermentations. Here, we present a syngas fermentation platform with a focus on safety installations and process analytical technology (PAT) that serves as a basis to assess the physiology of the PHB‐producing bacterium Rhodospirillum rubrum. The platform includes (i) off‐gas analysis with an online quadrupole mass spectrometer to measure CO consumption and production rates of H2 and CO2, (ii) an at‐line flow cytometer to determine the total cell count and the intracellular PHB content and (iii) different online sensors, notably a redox sensor that is important to confirm that the culture conditions are suitable for the CO metabolization of R. rubrum. Furthermore, we present as first applications of the platform a fed‐batch and a chemostat process with R. rubrum for PHB production from syngas.