1
Strategies for Continuous Processing in Microbial Systems

Julian Kopp1, Christoph Slouka2, Frank Delvigne3, and Christoph Herwig1,2

1Vienna University of Technology, Institute of Chemical, Environmental and Biological Engineering, Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, 1060, Vienna, Austria

2Vienna University of Technology, Institute of Chemical Environmental and Bioscience Engineering, Research Division Biochemical Engineering, Department of Chemical, Environmental and Biological Engineering, Gumpendorferstr. 1a, 1060, Vienna, Austria

3University of Liège, Terra Research and Teaching Center, Microbial Processes and Interactions (MiPI), Gembloux Agro-Bio Tech, Département GxABT, Bât. ABT09 G140 - Microbial, food and biobased technologies, Avenue de la Faculté d'Agronomie 2B, 5030, Gembloux, Belgium

1.1 Introduction

1.1.1 Microbial Hosts and Their Applications in Biotechnology

With regard to microbial cultivation technology, first associations might be drawn between classical food technological applications like ethanol fermentation in beer and wine and production of dry yeast for baking dough. Nevertheless, microbial systems play a fundamental role in all parts of biotechnology in a multitude of industrially used processes. Table 1.1 gives a - certainly not complete - list for possible application of microbes in today's industrial biotechnology.

There is a high variety of possible applications for a high number of different microorganisms (MOs) as shown in Table 1.1. There are classical working horses like Escherichia coli, Saccharomyces cerevisiae, and Bacillus spp. that can be cultivated easily to high cell densities and produce high amounts of the desired product. Other applications and microorganism suffer from inhibitory effects (e.g. inhibition from contaminants in waste water) and low biomass and product yields. Continuous cultivations are referred to increase the time-space yield (TSY) of many processes and provide optimal usage of installed assets. Still, most these processes are established for biomass generation or detoxification. Only very few continuously operated processes involve the production of recombinant compounds. The benefits and drawbacks of continuous cultivation will be discussed throughout this book chapter, focusing especially on microbial hosts. Hence, the ideal cultivation mode must be chosen wisely.

Table 1.1 Applications of microbial biotechnology.

Microbes Benefit Application in biotechnology Cultivation mode Source Aspergillus niger, Enterobacteria Overproduction of raw chemical by MOs, e.g. citric acid, lactic acid, vitamins Bulk chemicals Batch, fed-batch, and continuous cultivations [1-3] Thermophilic microbes - genera Picrophilus, Thermoplasma, Sulfolobus High-temperature stable enzymes Food, feed, textile, chemical, pharmaceutical, and other industrial sectors Continuous cultivation [4, 5] Thiobacillus/Leptospirillum Noble metal recovery Bio-oxidation Bioleaching [6] High diverse group, e.g. R. eutropha Conversion of toxic organic compounds, surface binding of heavy metals Bioremediation Batch and continuous processing [7, 8] E. coli, Bacillus, S. cerevisiae, P. pastoris Drug production, antibiotics, etc. Biopharmaceutical industry, enzyme industry, agricultural industry Fed-batch technology [9, 10] Lactobacillus and Bifidobacterium Functional food Probiotics Batch cultivation [11] S. cerevisiae, Zymomonas mobilis, Klebsiella oxytoca, Streptococcus fragilis Biomass fuels based on waste streams Biofuels Batch and continuous cultivations [12, 13] Wild type: Ralstonia eutropha, Alcaligenes latus; Recombinant: Aeromonas hydrophila, E. coli Photosynthetic: Synechocystis sp. Environmentally friendly non-petrochemical-based plastics Bioplastics (polyhydroxyalkanoates) Batch and fed-batch cultivation [14, 15] Haloferax mediterranei, other halophiles Tolerate high salt concentrations Detoxification in chemical waste streams Continuous cultivation [16, 17] High diverse groups - depending on application Waste to value PHA production; enzymes/organic acids Batch and fed-batch cultivations [18, 19] Mixed cultures, e.g. Proteus vulgaris, Rhodoferax ferrireducens, Geobacter sulfurreducens Energy generation from waste Microbial fuel cells Batch and continuous cultivations [20]

1.1.2 Regulatory Demands for Their Applied Cultivation Mode

The batch definitions in continuous manufacturing, preciously defined for mammalian cultivations, apply for microbial processes as well: "A Batch means a specific quantity of a drug or other material that is intended to have uniform character and quality, within specified limits, and is produced according to a single manufacturing order during the same cycle of manufacture. In the case of a drug product manufactured by a continuous process, it is a specific identified amount produced in a unit of time or quantity in a manner that assures its having uniform character and quality within specified limits" 21 CFR 210.3 2, or "a batch may correspond to a defined fraction of the production. The batch size can be defined either by a fixed quantity or by the amount produced in a fixed time interval" EU GMP Guide, Part II (ICH Q7).

More important than batch definition is the application of the quality-by-design (QbD) context to continuous processing. Generally, QbD mainly urges to relate critical quality attributes (CQAs) to critical process parameters (CPPs) and raw material attributes (RMA) to form a design space [21]: "A multidimensional combination and interaction of input variables and process parameters that have been demonstrated to provide assurance of quality of the product" for demonstrating process understanding. As proposed by current validation guidelines [22], stage 1 validation includes the execution of process characterization studies (PCS), which is the "collection and evaluation of data, from the process design stage throughout production. This establishes scientific evidence that a process is capable of consistently delivering quality product." PCS finally leads to the awareness of the mutual interplay of CPPs on CQAs. This demonstrates process robustness within multivariate normal operating ranges (NOR) and therefore finally proposes the control strategy including process and analytical controls. Currently, this is achieved by fusing development and manufacturing data.

Using an enhanced PCS approach, the determination of appropriate material specifications and process parameter ranges could follow a sequence such as the following [23]:

1. Identify potential sources of process variability.
2. Identify the material attributes and process parameters likely to have the greatest impact on drug substance quality.
3. Design and conduct studies (e.g. mechanistic and/or kinetic evaluations, multivariate design of experiments, simulations, modeling) to identify and confirm the links and relationships of material attributes and process parameters to drug substance CQAs.
4. Analyze and assess the data to establish appropriate ranges, including the establishment of a design space.

Even more, continuous processes require a different level of process understanding: as an example, classical recombinant protein production (RPP) using E. coli as a host pools the product solution after four days of processing. The time-variant dependency of CPPs and CQAs is finally integrated in one analytical result, and the process is also registered as such. Hence, batch processes are characterized by operating subsequent steps on the integral outcome of the current process step. Implementing continuous operations, we must understand the time dependency between CPPs and CQAs with the goal to have a time-invariant CQA process result. Hence, as microbial processes are more dynamic in terms of kinetics and stoichiometry, proper understanding of a dynamic design space and...