Klaus Joeris

In-situ microscopy: Online process monitoring of mammalian cell cultures

The in-situ microscope is a system developed to acquire images of mammalian cells directly inside a bioreactor (in-situ) duringa fermentation process. It requires only minimal operator intervention and it is well suited for either batch or long-termperfusion fermentation runs. The system fits into a 25 mm standard port and has a retractable housing, similar to the industry standard InTrac. Therefore, it can be cleaned and serviced without interruption of the process or risking contamination. A sampling zone inside the bioreactor encloses adefined volume of culture and an image sequence is taken. The height of the sampling zone is set by the control program and canbe adjusted during the cultivation to accommodate a wide range of change in cell density. The system has an infinity correctedoptical train and uses a progressive scan CCD camera to acquirehigh quality images. Process relevant information like cell density is extracted fromthe images by digital image processing software, currently in development for mammalian cells (CHO, BHK). The first version ofthe software will be able to estimate the cell density, cellsize distribution and to give information of the degree of aggregation (single and double cells, cell clusters).

Logistic Equations Effectively Model Mammalian Cell Batch and Fed‐Batch Kinetics by Logically Constraining the Fit

A four‐parameter logistic equation was used to fit batch and fed‐batch time profiles of viable cell density in order to estimate net growth rates from the inoculation through the cell death phase. Reduced three‐parameter forms were used for nutrient uptake and metabolite/product formation rate calculations. These logistic equations constrained the fits to expected general concentration trends, either increasing followed by decreasing (four‐parameter) or monotonic (three‐parameter). The applicability of this approach was first verified for Chinese hamster ovary (CHO) cells cultivated in 15‐L batch bioreactors. Cell density, metabolite, and nutrient concentrations were monitored over time and used to estimate the logistic parameters by nonlinear least squares. The logistic models fit the experimental data well, supporting the validity of this approach. Further evidence to this effect was obtained by applying the technique to three previously published batch studies for baby hamster kidney (BHK) and hybridoma cells in bioreactors ranging from 100 mL to 300 L. In 27 of the 30 batch data sets examined, the logistic models provided a statistically superior description of the experimental data than polynomial fitting. Two fed‐batch experiments with hybridoma and CHO cells in benchtop bioreactors were also examined, and the logistic fits provided good representations of the experimental data in all 25 data sets. From a computational standpoint, this approach was simpler than classical approaches involving Monod‐type kinetics. Since the logistic equations were analytically differentiable, specific rates could be readily estimated. Overall, the advantages of the logistic modeling approach should make it an attractive option for effectively estimating specific rates from batch and fed‐batch cultures.

Optical Inline Measurement Procedures for Counting and Sizing Cells in Bioprocess Technology

To observe and control cultivation processes, optical sensors are used increasingly. Important parameters for controlling such processes are cell count, cell size distribution, and the morphology of cells. Among turbidity measurement methods, imaging procedures are applied for determining these process parameters. A disadvantage of most previously developed imaging procedures is that they are only available offline which requires sampling. On the other hand, available imaging inline probes can so far only deliver a limited number of process parameters. This chapter presents new optical procedures for the inline determination of cell count, cell size distribution, and other parameters. In particular, by in situ microscopy an imaging procedure will be described which allows the determination of direct and nondirect cell parameters in real time without sampling.

A simple method for the isolation and purification of L-asparaginase from Enterobacter aerogenes.

Abstractl-Asparaginase fromEnterobacter aerogenes was purified by a simple method involving sonication of the crude cell mass, gel filtration with Sephacryl S-100 as the separating material, followed by ultrafiltration. Recent methods involve complex purification procedures of 5–6 steps. The isolation process resulted in 10-fold purification of the enzyme with a specific activity of 55 IU/mg protein and recovery of 54%. The purity was tested by capillary electrophoresis, used for the first time for documenting the purification ofl-asparaginase. The choice of the column material was critical in the purification process.

The Use of a Mobile Robot for Complete Sample Management in a Cell Culture Pilot Plant

A mobile robot system was developed that is capable of automating the complete sample management in a biotechnological laboratory. The robot consists of a wheeled platform and a mounted industrial robot arm with a gripper tool attached to it. The proper interaction with the biotechnological devices is given by the use of a colour camera for object recognition, a force/torque sensor to prevent damages and laser scanners for localisation. Furthermore necessary changes to the environment of the robot are kept to a minimum. By providing a scripting language, the robot system can be easily adapted to new devices and further tasks. By the use of this autonomous robot operating distinct devices, a fully automated sample management system was established which is available day and night.

Three-Dimensional Shape Estimation of BHK Cell Clusters from a Still Image Based on Shape from Shading for In-SITU Microscopy

In this contribution, the Lee and Rosenfelds local shape from shading (SFS) algorithm, the Tsai and Shahs linear SFS algorithm and the Bichsel and Pentlands propagation SFS algorithm are investigated with the aim of selecting the most suitable for three-dimensional shape estimation of clusters of mammalian baby hamster kidney cells (BHK cells) from an intensity image captured by an in-situ microscope in an industrial mammalian cell culture process. All three were implemented and tested using several thousand intensity images captured under varying experimental conditions. The Bichsel and Pentlands SFS algorithm was finally selected as the most suitable algorithm for three-dimensional shape estimation of BHK cell clusters. It is fast and provides less noise and more detailed depth estimates and therefore the best overall performance

Image Analysis Based Real Time-Control of Glucose Concentration

In-situ microscopy is the concept of monitoring micro organisms in the original production environment inside a fermentor. A new type of in-situ microscope suitable for both batch and long-term fermentation runs is presented. First results with the new type of in-situ microscope show that it is possible to integrate the in-situ microscopy system for process control purposes.

A novel in situ probe for oxygen uptake rate measurement in mammalian cell cultures

The newly developed in situ oxygen uptake rate (in situ OUR) probe presented in this article is based on the in situ microscope technology platform. It is designed to measure the oxygen uptake rate (OUR) of mammalian cells, an important parameter for metabolic flux analysis, inside a reactor (in situ) and in real‐time. The system isolates a known volume of cell culture from the bulk inside the bioreactor, monitors the oxygen consumption over time, and releases the sample again. The sample is mixed during the measurement with a new agitation system to keep the cells in suspension and prevent oxygen concentration gradients. The OUR measurement system also doubles as a standard dissolved oxygen (DO) probe for process monitoring when it is not performing OUR measurements. It can be equipped with two different types of optical sensors (i.e., DO, pH) simultaneously or a conventional polarographic DO‐probe (Clark type). This new probe was successfully tested in baby hamster kidney perfusion cell cultures.

Oxygen Uptake Rate (OUR) Estimation in High Density Mammalian Cell Perfusion Cultures

Mammalian cells are being widely used for the production of therapeu- tic proteins given their ability to correctly fold and glycosylate large molecules. However, productivities from a typical mammalian cell reactor are low and one approach to increase productivity is using perfusion systems for high cell density cell cultivations. Perfusion bioreactors are characterized by a high degree of com- plexity and require considerable effort to maintain. Due to the long duration of this type of process, disturbances in bioreactor operation can directly affect cellular metabolism and productivity. One parameter which has been shown to correlate to these metabolic shifts is the oxygen uptake rate (OUR).We present a method for real-time estimation of OUR in high density mammalian cell perfusion cultures. These computations are based on a global mass balance which does not require kLa data or reactor perturbations. The applicability of this approach was tested in a long-term BHK cell cultivation. Furthermore, real-time OUR data enables real-time metabolic flux estimation and cell physiological state monitoring.

INTEGRATING DISPARATE ANALYTICAL INSTRUMENTATION INTO AN AUTOMATED PROCESS CONTROL SYSTEM USED IN CELL CULTURE PROCESS DEVELOPMENT

Abstract This case study of a cell culture process development lab shows how various common analytical instruments were quickly and effectively integrated into a comprehensive process control and data management system. Streaming time-stamped results from these instruments directly into the process control system enabled automated real-time feedback and advanced characterizations of cell viability, instead of being limited to off-line, post-run analysis. Under the Ferm Works process control and data management software, drivers for instruments such as Cedex Cell Analyzer, YSI 2700 Biochemistry Analyzer, Bayer Blood Gas Analyzer and several other custom-made analytical instruments were easily added to the existing network of bioreactors and their hardware controllers (DCUs).