Analysis of Aggregates and Particles in Protein Pharmaceuticals E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 795

Veröffentlichungsjahr: 2011

Beschreibung

This book describes how to address the analysis of aggregates and particles in protein pharmaceuticals, provides a comprehensive overview of current methods and integrated approaches used to quantify and characterize aggregates and particles, and discusses regulatory requirements. Analytical methods covered in the book include separation, light scattering, microscopy, and spectroscopy.

Leseprobe

Table of Contents

Title Page

Copyright

Dedication Page

Contributors

Preface

Glossary

Chapter 1: The Critical Need for Robust Assays for Quantitation and Characterization of Aggregates of Therapeutic Proteins

1.1 Introduction

1.2 Methods for Sizing and Quantifying Soluble Aggregates

1.3 Methods for Sizing and Quantifying Particles

1.4 Methods for Characterizing Conformation of Protein Molecules in Aggregates

1.5 Regulatory Issues

1.6 Importance of the Current Book

References

Part 1: Methods Used for Detecting, Quantifying, and Sizing of Protein Aggregates and Particles

Chapter 2: Separation-Based Analytical Methods for Measuring Protein Aggregation

2.1 Introduction

2.2 Size Exclusion Chromatography

2.3 Analytical Ultracentrifugation

2.4 Field-Flow Fractionation

2.5 Electrophoresis

2.6 Other Potential Technologies

2.7 Summary

References

Chapter 3: Laser Light Scattering-Based Techniques Used for the Characterization of Protein Therapeutics

3.1 Introduction

3.2 Static Light Scattering

3.3 Dynamic Light Scattering

3.4 Case Studies and Drawbacks of Light Scattering-Based Methods

3.5 Conclusions and Discussion

References

Chapter 4: Online Detection Methods and Emerging Techniques for Soluble Aggregates in Protein Pharmaceuticals

4.1 Introduction

4.2 Online Methods for Soluble Aggregate Detection

4.3 Emerging Techniques and Novel Methods for Soluble Aggregate Detection

4.4 Summary

References

Chapter 5: Analytical Methods to Measure Subvisible Particulates

5.1 Introduction

5.2 USP < 788 > -Based Compendial Testing of Subvisible Particles

5.3 Other Techniques for Subvisible Particle Measurements

5.4 Potential Uses of These Methods

5.5 Case Studies

5.6 Summary and Future Directions

References

Chapter 6: Detection of Visible Particles in Parenteral Products

6.1 Introduction

6.2 Current Regulatory Requirements and Expectations

6.3 Sources of Visible Particulates

6.4 Clinical Concerns with Visible Particulates

6.5 Detection of Visible Particles

6.6 Process of Visible Particle Inspection

6.7 Case Studies

6.8 Summary

References

Chapter 7: Characterization of Aggregates and Particles Using Emerging Techniques

7.1 Introduction

7.2 Macro-IMS for Characterization of Proteins and Protein Aggregates

7.3 Flow Microscopy for Characterization of Protein Aggregates and Particles

7.4 Nanoparticle Tracking Analysis for Characterization of Protein Aggregates and Particles

7.5 Other Emerging Techniques

7.6 Summary

References

Part 2: Methods Used to Characterize Protein Aggregates and Particles

Chapter 8: Ultraviolet Absorption Spectroscopy

8.1 Introduction

8.2 Theoretical Background

8.3 Intrinsic Chromophores

8.4 Extrinsic Chromophores

8.5 Data Analysis

8.6 Difference Spectra

8.7 Indirect Measures of Protein Aggregation

8.8 Direct Measurement of Protein Aggregation

8.9 Utility of Extrinsic Dyes to Detect Protein Aggregation

8.10 Some Practical Considerations

8.11 Conclusions

References

Chapter 9: Fluorescence Spectroscopy to Characterize Protein Aggregates and Particles

9.1 Introduction

9.2 Introduction to Luminescence

9.3 Intrinsic Fluorescence

9.4 Use of Dyes (Extrinsic Fluorescence)

9.5 Steady-State Fluorescence

9.6 Time-Resolved Fluorescence

9.7 Fluorescence Quenching

9.8 Steady-State and Time-Resolved Fluorescence Anisotropy

9.9 Fluorescence Correlation Spectroscopy

9.10 Single-Particle Tracking

9.11 In Vivo Aggregate Characterization

9.12 Case Studies

9.13 Summary and Conclusions

References

Chapter 10: Infrared Spectroscopy to Characterize Protein Aggregates

10.1 Introduction

10.2 Infrared Spectroscopy—The Basics

10.3 Protein Structure and Infrared Spectroscopy

10.4 Protein Aggregation

10.5 Particles and Infrared Spectroscopy

10.6 Summary: Advantages and Disadvantages of IR Spectroscopic Analysis of Proteins, Protein Aggregates, and Particulate Material

References

Chapter 11: Raman Microscopy for Characterization of Particles

11.1 Introduction

11.2 Strengths and Weaknesses of Different Methods for Particle Identification

11.3 Experimental Setup of Raman Microscopy

11.4 Use of Raman Spectroscopy in Parenterals

11.5 Summary

References

Chapter 12: Microscopic Methods for Particle Characterization in Protein Pharmaceuticals

12.1 Introduction

12.2 Light Microscopy (LM)

12.3 Electron Microscopy (EM)

12.4 Atomic Force Microscopy (AFM)

12.5 Emerging Microscopic Technologies

12.6 Infrared (IR) Microscopy

12.7 Conclusions

References

Part 3: Integrated Approaches to Protein Aggregation and Particles

Chapter 13: Comparison of Methods for Soluble Aggregate Detection and Size Characterization

13.1 Introduction

13.2 The Measurement Can Change the Sample

13.3 Strengths and Limitations of Various Aggregation Assays

13.4 Approaches to Assay Cross Validation or Cross Qualification

13.5 Aggregation Assays for Formulation Development and Comparability Protocols

13.6 Concluding Remarks

References

Chapter 14: Protein Purification and Its Relation to Protein Aggregation and Particles

14.1 Introduction

14.2 Downstream Concepts for the Purification of Therapeutic Proteins

14.3 Controlling and Monitoring the Aggregate Level of Therapeutic Proteins During Purification Processing

14.4 Summary: Strategies to Reduce Aggregates During DSP

References

Chapter 15: Formulation Development and Its Relation to Protein Aggregation and Particles

15.1 Introduction

15.2 Stability of Liquid Protein Pharmaceuticals During Static Storage

15.3 Mechanical Stress Stability During Formulation and Shipment

15.4 Stability During Freeze Thaw

15.5 Special Challenges in High Concentration Protein Formulations

15.6 Special Challenges in Dried Protein Pharmaceuticals

15.7 Summary

References

Index

Color Plates

Copyright © 2011 by John Wiley & Sons, Inc. All rights reserved

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Library of Congress Cataloging-in-Publication Data:

Analysis of aggregates and particles in protein pharmaceuticals / edited by Hanns-Christian Mahler, Wim Jiskoot.

p.; cm.

Includes bibliographical references.

ISBN 978-0-470-49718-0 (cloth)

I. Mahler, Hanns-Christian. II. Jiskoot, Wim.

[DNLM: 1. Pharmaceutical Preparations—analysis. 2. Chemistry Techniques,

Analytical—methods. 3. Proteins—pharmacokinetics. QV 25]

LC classification not assigned

615.1'9—dc23

2011026192

To my beloved son, Conrad Louis,

and my wonderful wife, Christiane

—Hanns-Christian Mahler

". . . a dream that became a reality and

spread throughout the stars"

—Capt. Kirk

Contributors

Markus Bluemel, Analytical Research and Development of the Process Science and Production Department, Novartis Pharma AG, Basel, Switzerland

Mariola Bozova, Analytical Research and Development of the Process Science and Production Department, Novartis Pharma AG, Basel, Switzerland

Kevin Braeckmans, Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Ghent, Belgium

Shawn Cao, Process and Product Development, Amgen Inc., Thousand Oaks, California, USA

John F. Carpenter, Department of Pharmaceutical Sciences, University of Colorado Denver, Aurora, Colorado, USA

Barry Cherney, Division of Therapeutic Proteins, U.S. Food and Drug Administration, Rockville, Maryland, USA

Tapan K. Das, BioTherapeutics Pharmaceutical Sciences, Pfizer, St. Louis, Missouri, USA

Barthélemy Demeule, Late Stage Pharmaceutical Development, Genentech, Inc., South San Francisco, California, USA

John den Engelsman, Analytical Development and Validation, Schering Plough Research Institute, Oss, The Netherlands

Manuel Diez, Analytical Research and Development of the Process Science and Production Department, Novartis Pharma AG, Basel, Switzerland

Reza Esfandiary, Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas, USA

Roberto Falkenstein, Bioprocess Development, Purification, Pharma Technical Development Biologics Europe, Roche Diagnostics GmbH, Penzberg, Germany

Stefan Fischer, Late-Stage Pharmaceutical and Processing Development, Pharmaceutical and Device Development, Pharma Technical Development Biologics Europe, F. Hoffmann-La Roche Ltd, Basel, Switzerland

Kurt Forrer, Analytical Research and Development of the Process Science and Production Department, Novartis Pharma AG, Basel, Switzerland

Wolfgang Friess, Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University Munich, Munich, Germany

Patrick Garidel, Pharmaceutical Development, Process Science/Biopharmaceuticals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany

Andrea Hawe, Division of Drug Delivery Technology, Biologics Formulation Group, Leiden University, Leiden, The Netherlands

Stefan Hepbildikler, Bioprocess Development, Purification, Pharma Technical Development Biologics Europe, Roche Diagnostics GmbH, Penzberg, Germany

Andrea Herre, Biopharmaceuticals, Quality Control and Materials Testing, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany

Yijia Jiang, Process and Product Development, Amgen Inc., Thousand Oaks, California, USA

Wim Jiskoot, Division of Drug Delivery Technology, Biologics Formulation Group, Leiden University, Leiden, The Netherlands

Lene Jørgensen, Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Copenhagen, Denmark

Fabian Kebbel, Pharmaceutical Development, Process Science/Biopharmaceuticals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany

Werner Kliche, Biopharmaceuticals, Quality Control and Materials Testing, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany

Atanas Koulov, Analytical Research and Development of the Process Science and Production Department, Novartis Pharma AG, Basel, Switzerland

Wolfgang Kuhne, Bioprocess Development, Purification, Pharma Technical Development Biologics Europe, Roche Diagnostics GmbH, Penzberg, Germany

Markus Lankers, rap.Id Particle Systems GmbH, Berlin, Germany

Thorsten Lemm, Bioprocess Development, Purification, Pharma Technical Development Biologics Europe, Roche Diagnostics GmbH, Penzberg, Germany

Jun Liu, Late Stage Pharmaceutical Development, Genentech, Inc., South San Francisco, California, USA

Hanns-Christian Mahler, Pharmaceutical and Device Development, Pharma Technical Development Biologics Europe, F. Hoffmann-La Roche Ltd, Basel, Switzerland

Charles Russell Middaugh, Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas, USA

Linda Narhi, Process and Product Development, Amgen Inc., Thousand Oaks, California, USA

John S. Philo, Alliance Protein Laboratories, Thousand Oaks, California, USA

Robert A. Poole, Division of Drug Delivery Technology, Biologics Formulation Group, Leiden University, Leiden, The Netherlands

Miriam Printz, Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University Munich, Munich, Germany

Rahul S. Rajan, Process and Product Development, Amgen Inc., Thousand Oaks, California, USA

Hans Rogl, Bioprocess Development, Purification, Pharma Technical Development Biologics Europe, Roche Diagnostics GmbH, Penzberg, Germany

Amy S. Rosenberg, Division of Therapeutic Proteins, U.S. Food and Drug Administration, Rockville, Maryland, USA

Eva Rosenberg, Bioprocess Development, Purification, Pharma Technical Development Biologics Europe, Roche Diagnostics GmbH, Penzberg, Germany

Steven J. Shire, Late Stage Pharmaceutical Development, Genentech, Inc., South San Francisco, California, USA

Ronald Smulders, Analytical Method and Validation, Merck Manufacturing Division, Oss, The Netherlands

Marco van de Weert, Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Copenhagen, Denmark

Oliver Valet, rap.Id Particle Systems GmbH, Berlin, Germany

Hans Vos, Biological and Sterile Product Development, Schering plough Research Institute, Oss, The Netherlands

Gerhard Winter, Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University Munich, Munich, Germany

Frank Zettl, Bioprocess Development, Purification, Pharma Technical Development Biologics Europe, Roche Diagnostics GmbH, Penzberg, Germany

Hui Zhao, Analytical Research and Development of the Process Science and Production Department, Novartis Pharma AG, Basel, Switzerland

Ralf Zippelius, Pharma Biotech Production, Roche Diagnostics GmbH, Penzberg, Germany

Preface

Protein pharmaceuticals are increasingly used to treat life-threatening and chronic diseases, such as several forms of cancer and inflammation, viral infections, metabolic disorders, and central nervous system diseases. The pharmaceutical quality of these important products is the key to their safety and efficacy. To assess and assure the high quality of protein pharmaceuticals during their development, production, and use, science- and risk-based comprehensive analytical and process strategies are required.

With respect to the quality of protein pharmaceuticals, especially protein aggregation, particles have recently received increased interest from industry, academia, and regulators as some aggregates may have biological consequences, such as immunogenicity, altered bioactivity, or altered pharmacokinetics. In a protein pharmaceutical, aggregates would include all protein assemblies larger than the smallest naturally occurring, active subunit (e.g., monomer). Protein aggregates can differ in structure, size, “solubility,” reversibility, and type of bond, as further explained in the Glossary. Particles are defined as undissolved species—other than gas bubbles—that are unintentionally present in the product. Particles can be subdivided according to their size and can be proteinaceous or nonproteinaceous (see Glossary).

This book covers a broad range of analytical methods to detect and characterize the entire spectrum of protein aggregates and particles that may be present in a protein pharmaceutical. Moreover, it provides examples of how these methods can be applied during process and formulation development. An introduction to the topic of aggregates and particles, especially with regard to their potential immunogenicity, is given in Chapter 1. Chapters 2–4 deal with analytical methods related to “soluble” aggregates, based on separation, light scattering, and some emerging techniques. Chapters 5–7 deal with so-called insoluble aggregates and particles. Methods to measure subvisible particles are discussed first, followed by a discussion on visible particles. Finally, some emerging methods to measure insoluble matter are discussed. Chapters 8–12 deal with spectroscopic techniques that may help to characterize aggregates and particles, to better elucidate their structure, as well as to identify and differentiate proteinaceous and nonproteinaceous particles. Specific chapters are devoted to UV–vis, fluorescence, infrared and Raman spectroscopy, and microscopic methods. In Chapter 13, various methods are discussed and compared in the overall context of aggregate analysis. Finally, Chapters 14 and 15 are dedicated to discussing approaches to tackle aggregates and particles encountered during protein purification and formulation development.

This book not only provides a comprehensive overview of methods to analyze protein aggregates and particles but also includes case studies to illustrate challenges in this area. Technical and nontechnical scientists from analytics, process development, formulation development, quality control and quality assurance, regulatory sciences, manufacturing and project management, as well as interested parties from industry, academia, and regulatory authorities will benefit from this book. In addition, the book can serve as reference for students in the field of protein pharmaceuticals.

Wim Jiskoot

Hanns-Christian Mahler

Leiden, The Netherlands

Basel, Switzerland

October 2011

Glossary

Protein Aggregates All protein assemblies larger (e.g., dimer, hexamer, … million-mer, …) than the smallest naturally occurring, active subunit (e.g., monomer). Protein aggregates can differ in

1. Structure (native, denatured, or partially denatured)

2. Size (small to large; nanometer to millimeter range)

3. “Solubility”1

a. Soluble aggregates are aggregate species that are small enough to enter the column of a chromatographic separation or that cannot be filtered or centrifuged with predefined separation processes

b. Insoluble aggregates are aggregate species that can be filtered or centrifuged and thus typically escape chromatographic separation without further sample preparation

4. Reversibility (i.e., irreversible aggregates vs reversible aggregates/self-association)

5. Type of bond: covalent (S–S mediated or non-S–S mediated) versus noncovalent aggregates.

Particles Undissolved species—other than gas bubbles—that are unintentionally present in the product. These may include foreign particles or protein particles.

Foreign Particles Particles that are not intrinsic to the product and therefore typically are not proteinaceous. Examples include particles derived from contaminations of primary packaging (e.g., glass particles in vials), insects or insect parts, and contaminations derived from manufacturing process (e.g., cellulose fiber from cleaning wipes, metal parts from pumps, etc.).

Protein Particles Protein aggregates that are sufficiently large to be detected “visually” or by using detection methods for “subvisible” particles. A protein particle may also be defined as a large insoluble aggregate.

Visible Particles Insoluble matter that can be visualized by respective inspection aids and means such as light, rotation, magnification lenses, and adequate background. Visibility may depend on a number of factors. Visibility can be in the range of about 75–150 µm (arbitrary boundary) and larger. Visible particles may include foreign particles or protein particles.

Subvisible Particles Insoluble matter that cannot be easily visualized, which falls into the category of “particles” (i.e., larger than may be 1 µm but not yet visible, i.e., 75–150 µm; arbitrary boundaries). Subvisible particles may include foreign particles or protein particles.

Submicrometer Particles Insoluble matter between about 0.1 and 1 µm (arbitrary boundaries). Submicrometer particles may include foreign particles or protein particles.

Note:

1 Categorizing an aggregate as being “soluble” or “insoluble” depends on the method and conditions used (e.g., chromatographic column, eluent, filter size, filter material, centrifugation time and speed, etc.)

Chapter 1

The Critical Need for Robust Assays for Quantitation and Characterization of Aggregates of Therapeutic Proteins

John F. Carpenter

Department of Pharmaceutical Sciences, University of Colorado Denver, Aurora, Colorado, USA

Barry Cherney and Amy S. Rosenberg

Division of Therapeutic Proteins, U.S. Food and Drug Administration, Rockville, Maryland, USA

1.1 Introduction

Since the commercialization of monoclonal antibodies and recombinant therapeutic proteins in the 1980s, millions of lives have been saved or improved by these unique medicines. As with all therapeutics, assurance of product quality is key to providing a consistent clinical performance related to both safety and effectiveness. These assurances are more challenging with therapeutic proteins than with small molecular entities because of their heightened susceptibility to degradation via physical or chemical means, the dependency for their activity on often complex three-dimensional conformation, the complicated manufacturing processes needed for their production, and their propensity to induce immune responses, relative to small molecular entities. Indeed, extensive development and formulation studies to obtain a product that has appropriate stability during production, shipping, storage, and delivery to the patients are undertaken for each protein therapeutic. Robust, high resolution analytical methods are essential to meet the requirement to ensure product quality and for the development of the appropriate means to stabilize the protein.

Degradation of therapeutic proteins by one or multiple means (e.g., heat, light, agitation, and long-term storage in aqueous solution) causes a loss of product quality and, critically, may cause adverse effects on safety and efficacy. Among degradation products of therapeutic proteins that have adverse effects on safety and efficacy are protein aggregates [1, 2]. Aggregates include assemblies of protein molecules ranging from dimers to those large enough (e.g., ≥ 0.5 μm) to be classified as subvisible particles to larger, visible particles. Typically, oligomeric protein aggregates that are small enough to remain in solution are referred to as soluble aggregates and/or high molecular weight species. Assemblies of protein molecules large enough to be pelleted during centrifugation or filtered out of solution are often termed insoluble aggregates. Aggregate assemblies large enough to be detected and quantified with particle-counting instruments are usually called subvisible particles. If they are large enough to be seen with the unaided eye, particles are referred to as visible.

There are many challenges regarding the choice of analytical methods for assessment of protein aggregates and evaluation of the data from such methods. First, the methods employed must cover the extremely large size range of aggregates, usually requiring several methods to provide rigorous data across this size range [3, 4]. Second, analysis of aggregates is challenging because the mass fraction of the aggregates in a sample may be extremely low (e.g., 0.1–1.0%), thus requiring highly sensitive methods. Third, the process of conducting the analysis may change the aggregate composition in a sample. For example, sample dilution during size-exclusion chromatography may cause disaggregation [3]. Thus, it absolutely essential to use orthogonal methods to assure the accuracy and robustness of a given analytical method for protein aggregates [5]. Orthogonal methods are those that use a different operating principle to obtain corroborating data on a given analyte. For example, sedimentation velocity analytical ultracentrifugation (SV-AUC) is routinely used to confirm results from size-exclusion chromatography [2–5].

Besides presenting challenges for analytical methods, aggregates pose a particular concern for patient safety in that they may be potent inducers of immune responses with varying manifestations [1, 2]. The spectrum of clinical effects induced by aggregates pertains to a multiplicity of factors including but not limited to their size and valency, whether the key epitopes in the protein are in the native state or degraded, and whether the therapeutic protein product has an endogenous counterpart or is a foreign protein [1, 2]. On one end of the spectrum are mild effects including minor alterations of pharmacokinetics, while on the other end of the spectrum are serious clinical effects including frank anaphylactic reactions (IgG or IgE mediated), neutralization of product activity with loss of efficacy, and, for therapeutic counterparts of endogenous proteins, neutralization of both product and endogenous counterpart [1, 2]. The latter may result in a factor or cellular deficiency caused by loss of activity of the endogenous protein, if it has a unique function. Immune responses triggered by aggregates may target aggregate-specific (i.e., denatured or cryptic) epitopes, which do not cross react on the native protein, such as was the case in older studies of human serum albumin, intravenous immunoglobulin, and human growth hormone [1, 2]. Alternatively, immune responses triggered by aggregates in which the native protein conformation is preserved may neutralize the critical native domains that mediate activity, as is the case for example, for a percentage of patients taking type I interferon therapy [1, 2].

Indeed, advantage has been taken of the capacity of a protein, in its native state, to elicit an immune response by constructing or formulating target proteins as particulates in vaccines. Typically, in vaccines, the particulates are formed by adsorbing the protein antigen onto the surface of another material such as a colloidal aluminum salt or other experimental microparticles [6]. Heterogeneous particles may also be present among protein aggregates in therapeutic protein products [6] arising from adsorption of proteins to particles originating from filling pumps, such as stainless steel particles, or from the container closure, such as glass particles [7–9].

There is a preponderance of data in the literature that indicate that aggregates are culprits in causing immune responses to protein therapeutics and vaccine antigens (reviewed in [1, 2]). Therefore, for protein therapeutics, it is critical to assure that aggregates are at the lowest level practical and to minimize, in particular, the higher MW aggregates with multiple repetitive units, implicated in immune response induction. Meeting this standard requires that manufacturing processes, formulations, storage and shipping conditions, and education of patients and medical personnel administering these drugs are optimized for minimizing aggregate formation and that there are proper, robust, and high resolution assays developed to quantify and characterize protein aggregates for each therapeutic protein.

Developing such assays properly requires substantial knowledge and expertise—with respect to the specific challenges of the assays as well as to the properties of the given protein—because each therapeutic protein has unique physicochemical properties giving rise to different degradation pathways that engender aggregation. For example, even minor sequence changes (e.g., point mutations) or chemical degradation of a few residues in therapeutic monoclonal antibodies can cause different stability and aggregation behaviors [10, 11]. For these reasons, it is generally not sufficient to rely on so-called platform analytical methods for a given class of products such as monoclonal antibodies. Of course, the experience and expertise gained with analytical method development for similar proteins can be extremely valuable in guiding work on a new therapeutic protein, and a standard algorithm to method development can guide and substantially shorten the development cycle. But ultimately, analytical methods for each protein product must be “customized” for that particular protein. Shortcuts could lead to problems with reliability and accuracy of analytical methods that could compromise product quality and patient safety.

Therefore, appropriate time and resource investment, in the areas of process understanding and validation, personnel training, equipment, facilities, raw materials qualification, and analytical methods development and validation are required because these are ultimately essential to development of successful manufacturing and commercialization processes. The main methods for analysis of protein aggregates, their development and applications, challenges with their implementation, and the critical technical issues affecting their performance are expertly discussed in this book. These methods fall into the broad categories described below.

1.2 Methods for Sizing and Quantifying Soluble Aggregates

The main analytical method used to quantify and size soluble aggregates is high performance size-exclusion chromatography (HP-SEC) [3–5]. This method is used to characterize protein aggregates during process development for bulk drug substance and for release and stability assessments for drug product as well as during formulation development. Therefore, it is critically important that the values generated by HP-SEC for a given therapeutic protein precisely and accurately reflect the actual values for aggregates in a sample. Meeting this goal is challenging because of the potential for aggregates to dissociate during HP-SEC runs and/or to adsorb to the column media [3–5]. Owing to these problems, orthogonal methods should be employed to assess and assure the accuracy of results from HP-SEC and to guide development of robust HP-SEC methods for a given protein [3–5]. Currently, SV-AUC is a method used for this purpose, but this method also has its own challenges for obtaining robust, reproducible results for protein aggregates. There are also efforts to develop field-flow fractionation as a method to quantify and size soluble aggregates but because of its own particular challenges, this approach has not yet been as widely adopted as SEC-HPLC and SV-AUC.

1.3 Methods for Sizing and Quantifying Particles

Protein aggregates that are large enough to be considered as particles often constitute a minute mass fraction of the protein molecules in the drug product. Therefore, typically, the amount of protein in particles cannot be quantified based on loss of monomer. Instead, the particles are counted and sized by methods such as light obscuration, microflow imaging, and Coulter counting

[12]. Each of these methods has its benefits and drawbacks, and there is a substantial amount of new research in this area [12]. Among the critical points is the need to differentiate between microparticles containing both foreign materials (and to identify the foreign material) and protein and those containing protein alone, as this is essential for identifying causative factors and precluding aggregate formation in subsequent lots. Of course, because proteins readily adsorb to foreign microparticles arising from materials involved in product manufacture and storage (e.g., steel particles from piston pumps and glass particles for storage vessels) [9], it is expected that essentially all particles analyzed will contain at least a fraction of protein molecules.

Special attention is given to visible particles because each lot of parenteral dosage forms (e.g., vials, syringes, or more rarely ampoules) is subjected to 100% visual inspection after manufacturing. This approach requires highly skilled and trained operators. There are now efforts to automate “visible inspection,” which presumably could increase throughput.

1.4 Methods for Characterizing Conformation of Protein Molecules in Aggregates

The conformation of protein molecules in aggregates can affect their biological activity as well as the consequences of immune responses directed to such of aggregates. Characterization of conformation of protein molecules in soluble aggregates can be studied in a mixture or after separation of a given aggregate population. Analysis by methods such as fluorescence spectroscopy (intrinsic, with fluorescence dyes or quenching), UV absorbance and near-UV circular dichroism spectroscopy, binding to conformationally dependent antibodies for tertiary structure, and infrared and far-UV circular dichroism spectroscopy for secondary structure should be considered. A potency assay for biological function may be used, although some proteins aggregated in their native state may lose the capacity to interact productively through their cognate receptors. For protein molecules in particles, in some cases, the particles can be separated from the solution and studied with these same spectroscopic methods [8].

1.5 Regulatory Issues

It is a general principle that product quality attributes that contribute to clinical safety and efficacy must be identified, their levels correlated with clinical experience in patient populations, and specifications set for them to ensure that a favorable clinical performance profile is maintained with each lot of product produced. Of course, consistent with the principles established in the “Quality by Design Initiative,” enhanced knowledge of the attribute's impact on the safety and efficacy profile of the product may allow more flexibility in setting specifications. Aggregates are considered a critical attribute in terms of their potential to elicit immune responses and affect product activity, enhancing or diminishing potency. Thus, even in phase I clinical trials, the aggregate content must be characterized and routinely measured for each lot by well-qualified assays and provisional specifications set.

Exploring process and formulation modifications to minimize protein aggregation is crucial during product development. Of course, for products that pose a higher risk to patient safety, such as those for which neutralizing antibodies can neutralize endogenous proteins with unique biological functions, special care must be taken at the earliest stages of product development to accurately detect protein aggregates and to minimize their formation. For licensed products, it is important to ensure that the preferred aggregate assay for routine aggregate assessment, such as HP-SEC, can detect all the aggregate species that are likely to be present in the product, based on a full understanding of the process as it affects aggregation and on product degradation pathways. Therefore, orthogonal methods such as SV-AUC, field-flow fractionation, or other potential methods should be used to verify that any method or set of methods proposed for routine detection of aggregate species is capable of detecting and quantifying the desired range of aggregate species. It may be necessary to evaluate the robustness of the HP-SEC assay by demonstrating its ability to detect all protein aggregates generated under relevant stress conditions. If so confirmed, then HP-SEC may be the sole tool utilized for aggregate detection for routine assessments. However, following significant changes in manufacture, as is routinely done for nearly all protein therapeutics in the course of development, a more extensive comparability study must be performed in which the critical product quality attributes of the post-manufacturing change product are compared with those of the pre-change product by using well-qualified and robust assays. In such cases, aggregate assessment may again warrant orthogonal techniques to evaluate the levels and types of aggregates present in the post-manufacturing change product.

Regarding particulate assessment, the light obscuration test, as defined by USP 30 monograph <788>, requires analysis of particles > 10 and > 25 μm, leaving a gap in assessment of particles in the 0.1–10 μm subvisible range [6]. Although light obscuration can be used to quantitate particles that are between 2 and 10 μm, other methods such as Resonant Mass Measurement or Nanoparticle Tracking Analysis are currently being developed and evaluated for quantitation of particles that are < 2.0 μm in size. The use of novel methods for evaluation of protein particles in the GMP environment will require a concerted effort.

1.6 Importance of the Current Book

Protein aggregates are a critically important class of degradation products in therapeutic proteins. Therefore, robust analytical methods for aggregates are essential for assuring the safety and efficacy of these products and for guiding their development. The current book is an invaluable resource for researchers and managers working on therapeutic proteins. It provides expert reviews of the state-of-the art for the range of analytical methods used for assessment of protein aggregates and the numerous challenges that are unique to each method. Furthermore, the book provides insight into the future of method development and regulatory issues for protein aggregates. With comprehensive coverage of the key issues, this book will be a critical reference for the field for many years.

References

1. Rosenberg AS. Effects of protein aggregates: an immunologic perspective. AAPS J 2006;8:E501–E507.

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Part 1

METHODS USED FOR DETECTING, QUANTIFYING, AND SIZING OF PROTEIN AGGREGATES AND PARTICLES

Chapter 2

Separation-Based Analytical Methods for Measuring Protein Aggregation

Jun Liu, Barthélemy Demeule, and Steven J. Shire

Late Stage Pharmaceutical Development, Genentech, Inc., South San Francisco, California, USA

2.1 Introduction

Aggregation is one of the common degradation routes of protein pharmaceuticals and widely recognized as a critical quality attribute owing to its potential impact on product quality, safety, and efficacy [13]. The term protein aggregates refers to a broad spectrum of diversified self-associated states of proteins. The mechanisms of protein aggregation are quite complicated, and aggregates can form through different pathways [4, 5]. Stressed conditions such as denaturants, organic solvents, high or low temperatures, agitation, freeze–thaw, cavitation, and low or high pH can cause structural alteration in proteins and result in aggregation [47]. In addition, chemical modifications, such as deamidation, isomerization, oxidation, fragmentation, and disulfide shuffling, have been shown to be related to protein aggregation in some cases [8]. Protein aggregates, particularly those formed by covalent linkages or strong noncovalent associations between unfolded molecules, are likely to be irreversible after simple dilution and are generally more stable than those formed by relatively weak noncovalent bonds. Stable aggregates are easier to detect as they are less likely to dissociate during the analytical process. Protein aggregates can also be formed because of reversible self-association of native molecules through noncovalent interactions, such as charge–charge, dipole–dipole, and hydrophobic interactions. The multivalent reversible self-association of proteins in their native form has been associated with an increase in solution viscosity, protein gelation, protein crystallization, and phase separation [913]. Although aggregates formed by proteins in their native conformation have the potential to generate more antidrug antibodies than those formed by unfolded proteins [14, 15], it should be noted that not all the aggregates are the same. Protein aggregates formed by weak reversible self-association can be easily dissociated in human serum owing to significant dilution. Even protein aggregates, which either slowly or never dissociate, may be perfectly safe and harmless. From a quality control point of view, these aggregates may have to be treated differently from those that may cause potential safety issues.