Therapeutic Proteins, 2005
Methods and Protocols
Methods in Molecular Biology Series, Vol. 308

With the recent completion of the sequencing of the human genome, it is widely anticipated that the number of potential new protein drugs and targets will escalate at an even greater rate than that observed in recent years. However, identification of a potential target is only part of the process in developing these new next generation protein-based ?drugs? that are increasingly being used to treat human disease. Once a potential protein drug has been identified, the next rate-limiting step on the road to development is the production of sufficient authentic material for testing, charact- ization, clinical trials, and so on. If a protein drug does actually make it through this lengthy and costly process, methodology that allows the production of the protein on a scale large enough to meet demand must be implemented. Furthermore, large-scale production must not compromise the authenticity of the final product. It is also nec- sary to have robust methods for the purification, characterization, viral inactivation and continued testing of the authenticity of the final protein product and to be able to formulate it in a manner that retains both its biological activity and lends itself to easy administration. Therapeutic Proteins: Methods and Protocols covers all aspects of protein drug production downstream of the discovery stage. This volume contains contributions from leaders in the field of therapeutic protein expression, purification, characterization, f- mulation, and viral inactivation.

Biopharmaceutical Proteins.- Expression of Antibody Fragments by Periplasmic Secretion in Escherichia coli.- Secretory Production of Therapeutic Proteins in Escherichia coli.- Expression of Recombinant LTB Protein in Marine vibrio VSP60.- Heterologous Gene Expression in Yeast.- Pharmaceutical Proteins From Methylotrophic Yeasts.- Expression of Human Papillomavirus Type 16 L1 in Baculovirus Expression Systems.- Large-Scale Transient Expression of Therapeutic Proteins in Mammalian Cells.- Site-Specific Integration for High-Level Protein Production in Mammalian Cells.- Production of Recombinant Therapeutic Proteins by Mammalian Cells in Suspension Culture.- Transgene Control Engineering in Mammalian Cells.- Fusion to Albumin as a Means to Slow the Clearance of Small Therapeutic Proteins Using the Pichia pastoris Expression System.- High-Throughput Recovery of Therapeutic Proteins from the Inclusion Bodies of Escherichia coli.- Isolation, Solubilization, Refolding, and Chromatographic Purification of Human Growth Hormone from Inclusion Bodies of Escherichia coli Cells.- Large-Scale Preparation of Factor VIIa from Human Plasma.- Purification of Clinical-Grade Monoclonal Antibodies by Chromatographic Methods.- Virus Elimination and Validation.- Virus Removal by Nanofiltration.- Determining Residual Host Cell Antigen Levels in Purified Recombinant Proteins by Slot Blot and Scanning Laser Densitometry.- Principles of Biopharmaceutical Protein Formulation.- Solid-State Protein Formulation.- Stabilization of Therapeutic Proteins by Chemical and Physical Methods.- Extraction and Characterization of Vaccine Antigens from Water-in-Oil Adjuvant Formulations.- Probing Reversible Self-Association of Therapeutic Proteins by Sedimentation Velocity in the Analytical Ultracentrifuge.- Biological Characterization of Pegylated Interferons.- Characterization of Interferon ?2B Pegylated via Carboxyalkylation.- Quantifying Recombinant Proteins and Their Degradation Products Using SDS-PAGE and Scanning Laser Densitometry.- Extraction and Denaturing Gel Electrophoretic Methodology for the Analysis of Yeast Proteins.- Characterization of Therapeutic Proteins by Membrane and In-Gel Tryptic Digestion.- Oligosaccharide Release and MALDI-TOF MS Analysis of N-Linked Carbohydrate Structures from Glycoproteins.- Capillary Electrophoresis of Carbohydrates Derivatized With Fluorophoric Compounds.- High-Throughput LC/MS Methodology for ?(1?3)Gal Determination of Recombinant Monoclonal Antibodies.- Carbohydrate Structural Characterization of Fas Ligand Inhibitory Protein.- Top-Down Characterization of Protein Pharmaceuticals by Liquid Chromatography/Mass Spectrometry.- Sample Preparation Procedures for High-Resolution Nuclear Magnetic Resonance Studies of Aqueous and Stabilized Solutions of Therapeutic Peptides.

Includes supplementary material: sn.pub/extras