Online monitoring of the cell-specific oxygen uptake rate with an in situ combi-sensor.

In a biotechnological process, standard monitored process variables are pH, partial oxygen pressure (pO2), and temperature. These process variables are important, but they do not give any information about the metabolic activity of the cell. The ISICOM is an in situ combi-sensor that is measuring the cell-specific oxygen uptake rate (qOUR) online. This variable allows a qualitative judgement of metabolic cell activity. The measuring principle of the ISICOM is based on a volume element enclosed into a small measuring chamber. Inside the measuring chamber, the pO2 and the scattered light is measured. Within a defined measuring interval, the chamber closes, and the oxygen supply for the cells is interrupted. The decreasing oxygen concentration is recorded by the pO2 optode. This measuring principle, known as the dynamic method, determines the oxygen uptake rate (OUR). Together with the scattered light signal, the cell concentration is estimated and the qOUR is available online. The design of the ISICOM is focused on functionality, sterility, long-term stability, and response time behavior so the sensor can be used in bioprocesses. With the ISICOM, measurement of online and in situ measurement of the OUR is possible. The OUR and qOUR online measurement of an animal cell batch cultivation is demonstrated, with maximum values of OUR = 2.5 mmol L-1 h-1 and a qOUR = 9.5 pmol cell-1 day-1. Information about limitation of the primary and secondary substrate is derived by the monitoring of the metabolic cell activity of bacteria and yeast cultivation processes. This sensor contributes to a higher process understanding by offering an online view on to the cell behavior. In the sense of process analytical technology (PAT), this important information is needed for bioprocesses to realize a knowledge base process control.

Charged aerosol detector HPLC as a characterization and quantification application of biopharmaceutically relevant polysialic acid from E. coli K1.

Polysialic acid (polySia) is widely investigated in various biopharmaceutical applications (e.g. treatment of inflammatory neurodegenerative diseases), whereby a certain polySia chain length with an average degree of polymerization 20 (polySia avDP20) shows most promising effects. In this study, a rapid analytical method using a HPLC and charged aerosol detector (CAD) for the direct chain length characterization of biopharmaceutically relevant polySia was developed. It was evaluated as a fast alternative to the commonly used 1,2-diamino-4,5-methylenedioxybenzene (DMB) HPLC application. In contrast to HPLC-FLD, the CAD-application provides the actual chain length of polySia within ∼3 h. The reliability of the HPLC-CAD was evaluated with a commercial reference sample of known chain length and biotechnologically produced LC polySia (long chain polySia with a DP ∼130). Moreover, HPLC-CAD was successfully applied in the direct detection of oligo- and polySia until DP ∼65 and can be used to monitor the thermal hydrolysis and subsequent chromatographic isolation of polySia avDP20 (average degree of polymerization 20) without DMB sample derivatization. In addition, CAD was successfully applied for polySia quantification using a modified elution gradient. It was tested as a fast alternative to commonly used thiobarbituric acid (TBA) assay. A differentiation between LC polySia and smaller, hydrolysed polySia chains was intended and possible. For LC polySia and polySia avDP20, a quadratic relation between polySia mass-concentration and CAD signal was observed. In case of LC polySia, a quadratic dependency with a determination coefficient of R2 = 0.99 in a broad concentration range between 0.025 and 15 mg mL-1 was determined. Quantification of polySia avDP20 was found to have quadratic dependency with a determination coefficient of R2 = 0.99 in a concentration range between 0.02 and 0.25 mg mL-1. The HPLC-CAD was tested for quantification with polySia references of known concentration and showed high accordance with a concentration deviation ≤6.7%. The CAD quantification method was also applied in the polySia avDP20 production process and was compared to the TBA assay. Results of a correlation plot showed a high determination coefficient of R2 = 0.98. Overall, HPLC-CAD analysis was successfully tested as a suitable characterization and quantification application in the biopharmaceutical production of polySia.

Single-use membrane adsorbers for endotoxin removal and purification of endogenous polysialic acid from Escherichia coli K1.

Polysialic acid (polySia) is a promising molecule for various medical applications (e.g., treatment of inflammatory neurodegenerative diseases). In this study a complete production process for human-identical α-(2,8)-linked polySia was developed using a disposable bioreactor for cultivation of Escherichia coli K1 and single-use membrane adsorbers for downstream processing (DSP). The cultivation process was optimized to minimize complex media components and a maturation process after cultivation was established. The maturation led to further product release from the cell surface into the supernatant. Afterwards DSP was established using sodium hydroxide treatment combined with anion exchange membrane adsorbers for endotoxin and DNA depletion. After downstream processing the final product had neither detectable protein nor DNA contamination. Endotoxin content was below 3 EU mg-1. Investigation of the maximal chain length showed no effect of the harsh sodium hydroxide treatment during DSP on the stability of the polySia. Maximal chain length was ∼98 degree of polymerization.

Polysialic acid production using Escherichia coli K1 in a disposable bag reactor.

Polysialic acid (polySia), consisting of α-(2,8)-linked N-acetylneuraminic acid monomers plays a crucial role in many biological processes. This study presents a novel process for the production of endogenous polySia using Escherichia coli K1 in a disposable bag reactor with wave-induced mixing. Disposable bag reactors provide easy and fast production in terms of regulatory requirements as GMP, flexibility, and can easily be adjusted to larger production capacities not only by scale up but also by parallelization. Due to the poor oxygen transfer rate compared to a stirred tank reactor, pure oxygen was added during the cultivation to avoid oxygen limitation. During the exponential growth phase the growth rate was 0.61 h-1. Investigation of stress-related product release from the cell surface showed no significant differences between the disposable bag reactor with wave-induced mixing and the stirred tank reactor. After batch cultivation a cell dry weight of 6.8 g L-1 and a polySia concentration of 245 mg L-1 were reached. The total protein concentration in the supernatant was 132 mg L-1. After efficient and time-saving downstream processing characterization of the final product showed a protein content of below 0.04 mgprotein/gpolySia and a maximal chain length of ∼90 degree of polymerization.

Additive manufactured customizable labware for biotechnological purposes.

Yet already developed in the 1980s, the rise of 3D printing technology did not start until the beginning of this millennium as important patents expired, which opened the technology to a whole new group of potential users. One of the first who used this manufacturing tool in biotechnology was Lücking et al. in 2012, demonstrating potential uses 1, 2. This study shows applications of custom-built 3D-printed parts for biotechnological experiments. It gives an overview about the objects' computer-aided design (CAD) followed by its manufacturing process and basic studies on the used printing material in terms of biocompatibility and manageability. Using the stereolithographic (SLA) 3D-printing technology, a customizable shake flask lid was developed, which was successfully used to perform a bacterial fed-batch shake flask cultivation. The lid provides Luer connectors and tube adapters, allowing both sampling and feeding without interrupting the process. In addition, the digital blueprint the lid is based on, is designed for a modular use and can be modified to fit specific needs. All connectors can be changed and substituted in this CAD software-based file. Hence, the lid can be used for other applications, as well. The used printing material was tested for biocompatibility and showed no toxic effects neither on mammalian, nor on bacteria cells. Furthermore an SDS-PAGE-comb was drawn and printed and its usability evaluated to demonstrate the usefulness of 3D printing for everyday labware. The used manufacturing technique for the comb (multi jet printing, MJP) generates highly smooth surfaces, allowing this application.

Sensors for disposable bioreactors.

Modern bioprocess monitoring demands sensors that provide on-line information about the process state. In particular, sensors for monitoring bioprocesses carried out in single-use bioreactors are needed because disposable systems are becoming increasingly important for biotechnological applications. Requirements for the sensors used in these single-use bioreactors are different than those used in classical reusable bioreactors. For example, long lifetime or resistance to steam and cleaning procedures are less crucial factors, while a requirement of sensors for disposable bioreactors is a cost that is reasonable on a per-use basis. Here, we present an overview of current and emerging sensors for single-use bioreactors, organized by the type of interface of the sensor systems to the bioreactor. A major focus is on non-invasive, in-situ sensors that are based on electromagnetic, semiconducting, optical, or ultrasonic measurements. In addition, new technologies like radio-frequency identification sensors or free-floating sensor spheres are presented. Notably, at this time there is no standard interface between single-use bioreactors and the sensors discussed here. In the future, manufacturers should address this shortcoming to promote single-use bioprocess monitoring and control.