Bioprocessing Technology for Production of Biopharmaceuticals and Bioproducts

List of Contributors xi
 
Part I Case Study 1
 
1 Bacillus and the Story of Protein Secretion and Production 3
Giulia Barbieri, Anthony Calabria, Gopal Chotani, and Eugenio Ferrari
 
1.1 Bacillus as a Production Host: Introduction and Historical Account 3
 
1.2 The Building of a Production Strain: Genetic Tools for B. subtilis Manipulation 5
 
1.2.1 Promoters 5
 
1.2.2 Vectors for Building a Production Strain 6
 
1.2.3 B. subtilis Competent Cell Transformation 7
 
1.2.4 Protoplasts-Mediated Manipulations 9
 
1.2.5 Genetics by Electroporation 9
 
1.3 B. subtilis Secretion Systemand Heterologous Protein Production 9
 
1.3.1 Bacillus Fermentation and Recovery of Industrial Enzyme 11
 
1.3.2 Fermentation Stoichiometry 12
 
1.3.3 Fermentor Kinetics and Outputs 14
 
1.3.4 Downstream Processing 17
 
1.4 Summary 21
 
References 21
 
2 New Expression Systems for GPCRs 29
Dimitra Gialama, Fragiskos N. Kolisis, and Georgios Skretas
 
2.1 Introduction 29
 
2.2 Recombinant GPCR Production - Traditional Approaches for Achieving High-Level Production 39
 
2.3 Engineered Expression Systems for GPCR Production 42
 
2.3.1 Bacteria 42
 
2.3.2 Yeasts 48
 
2.3.3 Insect Cells 51
 
2.3.4 Mammalian Cells 54
 
2.3.5 Transgenic Animals 54
 
2.3.6 Cell-Free Systems 56
 
2.4 Conclusion 57
 
References 58
 
3 Glycosylation 71
Maureen Spearman, Erika Lattová, Hélène Perreault, andMichael Butler
 
3.1 Introduction 71
 
3.2 Types of Glycosylation 72
 
3.2.1 N-linked Glycans 72
 
3.2.2 O-linked Glycans 74
 
3.3 Factors Affecting Glycosylation 76
 
3.3.1 Nutrient Depletion 76
 
3.3.2 Fed-batch Cultures and Supplements 79
 
3.3.3 Specific Culture Supplements 80
 
3.3.4 Ammonia 82
 
3.3.5 pH 82
 
3.3.6 Oxygen

CLAIRE KOMIVES, PHD, is a Professor in the Chemical and Materials Engineering Department at San Jose State University. Her research interests focus on the development of low cost snake antivenom compounds.
 
WEICHANG ZHOU, PHD, is Chief Technology Officer, Biologics Development & Manufacturing at WuXi Biologics. He has previously worked at Genzyme Corporation and Merck Research Laboratories.