Analysis of Aggregates and Particles in Protein Pharmaceuticals

Enables researchers to better detect, measure, and characterizeaggregates and particles during protein drug development

Featuring contributions from leading pharmaceuticalinvestigators around the world, this text enables researchers tochoose the most appropriate analytical methods in order to detectand characterize the full spectrum of protein aggregates andparticles that can be present in a protein pharmaceutical.Moreover, the authors provide detailed examples of how thesemethods can be applied during both process and formulationdevelopment, helping researchers better manage the problem ofprotein instability as they seek to develop protein drugs.

The text begins with an introduction to aggregates andparticles, focusing on their potential immunogenicity. Next, itcovers:

Methods to detect and measure soluble aggregates based onseparation, light scattering, and new technology

Methods to detect and measure insoluble aggregates and bothsub–visible and visible particles

Spectroscopic techniques such as UV/VIS, fluorescence, infraredand Raman spectroscopy, and microscopy to characterize aggregatesand particles, elucidate their structures, and identifyproteinaceous and non–proteinaceous particles

Comparative advantages and disadvantages of the methods forprotein aggregate analysis

Approaches for managing aggregates and particles found duringprotein purification and formulation development

Throughout the text, case studies help researchers deal with thechallenges that arise in detecting and analyzing protein aggregatesand particles.

Protein pharmaceuticals are increasingly used to treatlife–threatening and chronic diseases, including cancer, viralinfections, metabolic disorders, and central nervous systemdiseases. With Analysis of Aggregates and Particles in ProteinPharmaceuticals as their guide, pharmaceutical researchers nowhave an important tool in overcoming the challenging problem ofaggregates and particles in protein drug development.

Contributors ix

Preface xiii

Glossary xv

1 The Critical Need for Robust Assays for Quantitation andCharacterization of Aggregates of Therapeutic Proteins 1
John F. Carpenter, Barry Cherney, and Amy S. Rosenberg

I METHODS USED FOR DETECTING, QUANTIFYING, ANDSIZING OF PROTEIN AGGREGATES AND PARTICLES 9

2 Separation–Based Analytical Methods for Measuring ProteinAggregation 11
Jun Liu, Barthelemy Demeule, and Steven J. Shire

3 Laser Light Scattering–Based Techniques Used for theCharacterization of Protein Therapeutics 37
John den Engelsman, Fabian Kebbel, and Patrick Garidel

4 Online Detection Methods and Emerging Techniques for SolubleAggregates in Protein Pharmaceuticals 61
Tapan K. Das

5 Analytical Methods to Measure Subvisible Particulates 85
Shawn Cao, Linda Narhi, Yijia Jiang, and Rahul S. Rajan

6 Detection of Visible Particles in Parenteral Products117
Ronald Smulders, Hans Vos, and Hanns–Christian Mahler

7 Characterization of Aggregates and Particles Using EmergingTechniques 133
Hui Zhao, Manuel Diez, Atanas Koulov, Mariola Bozova, MarkusBluemel, and Kurt Forrer

II METHODS USED TO CHARACTERIZE PROTEIN AGGREGATES ANDPARTICLES 169

8 Ultraviolet Absorption Spectroscopy 171
Reza Esfandiary and Charles Russell Middaugh

9 Fluorescence Spectroscopy to Characterize Protein Aggregatesand Particles 201
Robert A. Poole, Andrea Hawe, Wim Jiskoot, and KevinBraeckmans

10 Infrared Spectroscopy to Characterize Protein Aggregates227
Marco van de Weert and Lene Jørgensen

11 Raman Microscopy for Characterization of Particles 249
Stefan Fischer, Oliver Valet, and Markus Lankers

12 Microscopic Methods for Particle Characterization in ProteinPharmaceuticals 269
Patrick Garidel, Andrea Herre, and Werner Kliche

III INTEGRATED APPROACHES TO PROTEIN AGGREGATION ANDPARTICLES 303

13 Comparison of Methods for Soluble Aggregate Detection andSize Characterization 305
John S. Philo

14 Protein Purification and Its Relation to Protein Aggregationand Particles 335
Roberto Falkenstein, Stefan Hepbildikler, Wolfgang Kuhne,Thorsten Lemm, Hans Rogl, Eva Rosenberg, Gerhard Winter, FrankZettl, and Ralf Zippelius

15 Formulation Development and Its Relation to ProteinAggregation and Particles 369
Miriam Printz and Wolfgang Friess

Index 389

HANNS–CHRISTIAN MAHLER is Head of Pharmaceutical and DeviceDevelopment, Pharma Technical Development Biologics Europe at F.Hoffmann–La Roche Ltd. He is also Lecturer at the Institute forPharmaceutical Technology at the University of Frankfurt/Main,Germany.

WIM JISKOOT is Professor and Head of the BiologicsFormulation Group in the Leiden/Amsterdam Center for Drug Research(LACDR) at Leiden University.