Biophysical characterization of proteins in developing biopharmaceuticals

Description

Biophysical Characterization of Proteins in Developing Biopharmaceuticals, Second Edition, presents the latest on the analysis and characterization of the higher-order structure (HOS) or conformation of protein based drugs. Starting from the very basics of protein structure, this book explains the best way to achieve this goal using key methods commonly employed in the biopharmaceutical industry. This book will help today's industrial scientists plan a career in this industry and successfully implement these biophysical methodologies. This updated edition has been fully revised, with new chapters focusing on the use of chromatography and electrophoresis and the biophysical characterization of very large biopharmaceuticals. In addition, best practices of applying statistical analysis to biophysical characterization data is included, along with practical issues associated with the concept of a biopharmaceutical's developability and the technical decision-making process needed when dealing with biophysical characterization data. Presents basic protein characterization methods and tools applicable to (bio)pharmaceutical research and development Highlights the capabilities and limitations of each technique Discusses the underlining science of each tool Empowers industrial biophysical chemists by providing a roadmap for applying biophysical tools Outlines the needs for new characterization and analytical tools in the biopharmaceutical industry Biophysical Characterization of Proteins in Developing Biopharmaceuticals Copyright Contributors Prefaces for the second edition List of abbreviations and symbols 1 - The complexity of protein structure and the challenges it poses in developing biopharmaceuticals 1.1 The basics of protein higher order structure (HOS) 1.1.1 The levels of protein HOS 1.1.2 Stabilizing the HOS of proteins 1.1.3 Dynamics properties of a Protein's HOS 1.1.4 Finer structural alteration of proteins 1.2 The search for how proteins attain their correct HOS: the protein folding problem 1.2.1 In vivo production of proteins: revisiting the protein folding problem 1.2.2 In vivo production of proteins: avoiding and eliminating folding errors via the use of chaperones 1.3 Surprises in the world of protein folding: intrinsically disordered or unstructured proteins (an apparent challenge to the ... 1.4 Proteins and the biopharmaceutical industry: problems and challenges 1.4.1 Impact of PTMs on the HOS of protein biopharmaceuticals 1.4.2 Impact of changes in noncovalent interactions (secondary bonds) on the HOS of protein biopharmaceuticals 1.4.3 A more detail discussion concerning protein biopharmaceutical aggregations and its influence on HOS 1.4.4 The novelty of different classes of protein biopharmaceuticals that create unique questions and challenges in characterizin ... 1.4.4.1 Example 1: Fc fusion proteins 1.4.4.2 Example 2: PEGylated proteins and antibody drug conjugates (ADCs) 1.4.4.3 Example 3: viruses, VLPs 1.5 Conclusion References Further reading 2 - Biophysical characterization and its role in the biopharmaceutical industry 2.1 Drug development process 2.2 Protein drugs (biopharmaceuticals) 2.3 The role of biophysical characterization in biopharmaceutical drug development 2.3.1 Biophysical properties: the developability issue at the research–development interphase 2.3.1.1 Limiting factors that influence the level of effort put into assessing a protein drug's developability 2.3.1.2 Tools and approaches in assessing developability of a protein drug at the research–development interface 2.3.2 Important early biophysical activities required after a protein drug transitions from research to development 2.3.3 Developing the biophysical higher order structure (HOS) fingerprint of a protein drug 2.3.3.1 Developing the HOS fingerprint of a protein drug using biophysical tools that provide indirect rather than direct structura ... 2.3.3.2 The widespread importance of biophysical characterization in biopharmaceutical comparability studies (stability, compatibil ... 2.3.3.3 The role of biophysical comparability in the biosimilar world 2.3.4 Understanding the impact of variant forms of a protein drug on its HOS and biophysical properties 2.4 The challenges in conducting biophysical measurements to detect changes in a protein drug's HOS 2.4.1 Molecular size and molecular heterogeneity (PTMs) challenge 2.4.2 Protein concentration challenge 2.4.3 Challenges arising from a biopharmaceutical's formulation 2.4.4 Container closure challenges 2.4.5 Challenges of sample measurement time versus sample stability 2.5 Regulatory needs and considerations References Further reading 3. Biopharmaceutical industry's biophysical toolbox 3.1 Attributes of a single biophysical tool to characterize and detect changes in the higher order structure of a biopharmaceutical 3.2 Studying the biophysical properties of a biopharmaceutical as an indirect approach for characterizing changes in its HOS 3.3 General considerations in analyzing the biophysical properties of biopharmaceuticals 3.3.1 Dealing with the physical and chemical state of a biopharmaceutical in conducting biophysical measurements (sample processing) 3.3.2 Biophysical properties of a biopharmaceutical that can be probed to assess information about its HOS 3.3.2.1 Changes in quaternary structure 3.3.2.2 Changes in secondary and tertiary structures 3.4 The utility of using stress to monitor changes in the HOS profile of a protein drug 3.5 Present biophysical toolbox 3.5.1 Methods for studying the hydrodynamic properties 3.5.2 Methods for studying the thermodynamic properties 3.5.3 Methods for studying chromatographic properties 3.5.4 Methods for studying electrophoretic properties 3.5.5 Methods for studying spectroscopic properties 3.5.6 Methods for studying the mass spectrometry properties 3.5.7 Methods for studying the LS properties 3.5.8 Methods for particle analysis 3.5.9 Developing a better biophysical toolbox 3.6 Conclusion References Further reading 4 - An introduction and hierarchical organization of the biophysical tool in section II 4.1 Introduction 4.2 The standard class of biophysical tools used in the biopharmaceutical industry 4.2.1 Tier 1 biophysical tools 4.2.2 Tier 2 biophysical tools 4.3 The advanced class of biophysical tools used in the biopharmaceutical industry 4.3.1 Tier 3 biophysical tools 4.3.2 Tier 4 biophysical tools 4.4 An overview of section II References Further reading 5 - UV-absorbance, fluorescence and FT-IR spectroscopy in biopharmaceutical development 5.1 Introduction 5.2 The origins of electronic absorption, fluorescence, and FT-IR spectroscopy 5.2.1 Protein far-UV absorption 5.2.2 Protein near-UV absorption 5.2.3 Fluorescence spectroscopy 5.2.4 FT-IR spectroscopy 5.3 Conformational analysis of proteins in solution 5.3.1 UV-derivative spectroscopy 5.3.2 FT-IR of proteins in solution 5.3.3 High concentration solutions 5.4 Optical and vibrational spectroscopy and product comparability 5.4.1 UV-absorption spectra for comparability 5.4.2 Intrinsic fluorescence spectra for comparability 5.4.3 FT-IR spectra for comparability 5.5 Optical and vibrational spectroscopy and high throughput methods 5.5.1 Extrinsic fluorescent probes 5.5.2 Differential scanning fluorimetry 5.5.3 High throughput applications of FT-IR 5.6 Solid-state measurements 5.6.1 FT-IR of proteins in the solid state 5.6.2 Solid-state protein fluorescence 5.7 Conclusions References 6 - Biopharmaceutical applications of protein characterisation by circular dichroism spectroscopy 6.1 Introduction 6.1.1 Theory 6.1.1.1 The physical origins of CD signals 6.1.1.1.1 Far-UV absorption of the peptide bond 6.1.1.1.2 Near-UV CD 6.1.1.1.3 Units and equations 6.2 Instrumentation 6.2.1 General setup of a bench-top instrument 6.3 Data generated 6.3.1 Types of data generated 6.3.2 Quantitative assessment of information content (as a function of wavelength) 6.4 Guide to collecting good data 6.4.1 Amount of time required to make measurements 6.4.2 Calibration schedule 6.4.2.1 Wavelength calibration 6.4.2.2 Calibration for magnitude and polarization 6.4.3 Data collection protocols 6.4.3.1 Protein concentration 6.4.3.2 Optical cells 6.4.3.2.1 Pathlength 6.4.3.2.2 Loading optical cells 6.4.3.2.3 Calibration of optical cells 6.4.3.3 Choice of sample conditions 6.4.3.4 Instrument settings 6.4.3.5 Sources of interference/troubleshooting 6.4.3.5.1 Signal-to-noise 6.4.3.5.2 HT level 6.4.3.6 Record/log keeping: essential details to note 6.4.3.7 Sources of errors in data collection 6.4.3.8 Special considerations for membrane proteins 6.5 Data processing and analyses 6.5.1 Data processing software—elements and procedures and identifying sources of error in the data 6.5.2 Secondary structure analyses 6.5.3 Accuracy: spectral data versus derived results (i.e., what types of samples cannot be accurately analyzed, but can be measu ... 6.5.4 Public repository of protein CD data (PCDDB): availability and uses 6.5.5 Data validation 6.6 Role in the research industry 6.6.1 CD data in the protein biopharmaceutical development process 6.6.1.1 Biosimilars 6.6.1.2 Glycosylation 6.6.2 CD spectroscopy and international regulatory bodies 6.7 Technology availability 6.7.1 Software 6.7.2 Commercial vendors and suppliers of accessories/key supplies 6.7.2.1 Instruments 6.7.2.2 Cells/calibration standards 6.8 Future developments 6.8.1 SRCD spectroscopy Acknowledgements References Further reading Books Websites General introductions to CD and secondary structure analyses Instruments, accessories and standards suppliers Online videos 7 - Size-exclusion chromatography (SEC) in biopharmaceutical process development 7.1 Introduction 7.2 Basic theory of normal or ideal SEC 7.3 Maximizing SEC separation by enhancing the usage of pore volume and pore structure 7.4 Characteristics of pore structure 7.5 Nonideal SEC chromatography 7.5.1 Attractive interactions—enthalpic effects 7.5.2 Repulsive interactions and hydrodynamic chromatography 7.6 Assessing and maintaining an optimum SEC chromatography method 7.7 Detectors 7.7.1 Ultraviolet 7.7.2 Fluorescence 7.7.3 Refractive index (via differential refractive index detector) 7.7.3.1 Unique troublesome properties of differential refractive index detectors 7.7.4 Light scattering 7.7.4.1 Unique and troublesome properties of light scattering detectors 7.7.5 Mass spectrometry 7.8 Multidetector SEC 7.8.1 Detecting conformational change and/or microheterogeneity by SEC 7.8.2 Critical assessment of multidetector SEC (inter-detector volume effect) 7.8.3 Protein extinction coefficient 7.8.4 Characterizing the concentration-dependent behavior of biopharmaceuticals 7.9 Aggregation 7.10 Technology advances 7.11 Conclusion References 8 - Scattering techniques for the characterization of biopharmaceuticals 8.1 Introduction 8.2 Intensity- and time-dependent light scattering 8.2.1 Static light scattering (SLS) 8.2.1.1 SLS aggregation 8.2.1.2 SLS: size 8.2.1.3 SLS: concentration 8.2.2 Dynamic light scattering (DLS) 8.2.2.1 DLS: aggregation 8.2.2.2 DLS: concentration 8.2.2.3 DLS: viscosity 8.3 General comment concerning SLS and DLS 8.4 The “dust problem” in SLS and DLS 8.5 X-ray scattering: characterization of proteins in solution using small-angle X-ray scattering 8.5.1 Physical basis of the method 8.5.2 Information generated by the method 8.5.2.1 Radius of gyration 8.5.2.2 Aggregation 8.5.2.3 High concentration solutions 8.5.2.4 Oligomeric state 8.5.2.5 Flexibility and the Kratky plot 8.5.2.6 P(r)—Pair-distribution function 8.5.2.7 Calculation of I(q) from atomic coordinate sets 8.5.2.8 Low-resolution molecular envelopes 8.5.2.9 Retrospective 8.5.2.10 Structural fluctuations 8.5.3 Method requirements and performance 8.5.3.1 Sample preparation 8.5.3.2 Data collection 8.5.3.3 Data processing (procedures) 8.5.3.3.1 Computer software availability, limitations 8.5.3.3.2 SEC-SAXS 8.5.3.3.3 Statistical analysis procedures to assess level of uncertainty in information, especially in terms of assessing differences ... 8.5.4 Role in research versus process development 8.5.4.1 Differentiate when data is nice to have versus must have (for comparability study, filing, etc.) 8.5.5 Technology update: recent and future advances and unique applications 8.5.5.1 Commercial vendors and suppliers of accessories/key supplies 8.5.5.2 Comments on future development and what May Be required to improve the methodology References 9. Characterizing biopharmaceuticals using analytical ultracentrifugation 9.1 Introduction 9.2 Unique features of the analytical ultracentrifuge that make it different from other centrifuges 9.3 Theory 9.4 Utility of AUC in the biopharmaceutical industry 9.5 Boundary SV-AUC 9.5.1 Boundary SV-AUC on a homogeneous protein sample—measuring a protein's sedimentation coefficient in the absence of diffusion ... 9.5.2 Boundary SV-AUC on a homogeneous protein sample—measuring a protein's sedimentation coefficient in the presence of diffusio ... 9.5.3 Assessing the homogeneity of protein biopharmaceuticals: the influence of various forms of sample heterogeneity on boundary ... 9.5.4 Determining a sample's distribution of sedimentation coefficients in order to assess its level of heterogeneity via boundar ... 9.5.5 Determining a sample's distribution of sedimentation coefficients in order to assess its level of heterogeneity via boundar ... 9.5.6 Assessing aggregation via boundary SV-AUC 9.5.6.1 Limit of quantitation (LOQ) in measuring aggregation 9.5.6.2 Detecting aggregation involving reversible concentration-dependent self-association 9.6 Expanding the dynamic size range that can be characterized in a single SV-AUC run 9.7 Band SV-AUC 9.8 Sedimentation equilibrium, SE-AUC 9.8.1 Assessing stable noninteracting aggregates versus aggregation involving reversible concentration-dependent self-association 9.8.2 Qualitative assessment of aggregation at very high protein concentrations 9.9 Density-gradient SE-AUC 9.10 AUC detectors 9.10.1 Variable wavelength absorbance detector 9.10.2 Refractometric detection using the Rayleigh interferometer 9.10.2.1 Use of the Rayleigh interferometer to experimentally assess the extinction coefficient of protein biopharmaceuticals 9.10.2.2 Difference SV-AUC using the Rayleigh interferometer: increasing the ability to assess changes in the HOS of protein biophar ... 9.10.3 Fluorescence detection using the AU-FDS 9.11 Miscellaneous helpful information about conducting AUC experiments 9.12 The introduction of the next (third) generation analytical ultracentrifuge (Optima) by Beckman Coulter 9.12.1 Performance issues in the initially-released third generation analytical ultracentrifuge (Optima) by Beckman Coulter 9.12.2 Recommended end user QC testing of the Optima 9.12.2.1 Initial Optima installation and long-term performance testing 9.12.2.2 Possibility for Optima users to qualitatively detect initial quality issues of AUC cell hardware (centerpiece and cell hous ... 9.13 Conclusions References Further reading 10 - Submicrometer, micrometer and visible particle analysis in biopharmaceutical research and development 10.1 Introduction 10.2 Overview of analytical methods 10.2.1 Dynamic light scattering 10.2.1.1 Underlying principle and readout parameters 10.2.1.2 Application of DLS during biopharmaceutical development 10.2.1.3 Considerations for DLS measurements 10.2.1.4 Instruments and new trends 10.2.2 Nanoparticle tracking analysis 10.2.2.1 Underlying principle and readout parameters 10.2.2.2 Application of NTA during biopharmaceutical development 10.2.2.3 Considerations for NTA measurements 10.2.2.4 Instruments and new trends 10.2.3 Asymmetrical flow field flow fractionation 10.2.3.1 Underlying principle and readout parameters 10.2.3.2 Application of AF4 during biopharmaceutical development 10.2.3.3 Considerations for AF4 measurements 10.2.3.4 Instruments and new trends 10.2.4 Resonant mass measurement 10.2.4.1 Underlying principle and readout parameters 10.2.4.2 Application of RMM during biopharmaceutical development 10.2.4.3 Considerations for RMM measurements 10.2.4.4 Instruments and new trends 10.2.5 Electrical sensing zone method 10.2.5.1 Underlying principle and readout parameters 10.2.5.2 Application of ESZ during biopharmaceutical development 10.2.5.3 Considerations for ESZ measurements 10.2.5.4 Instruments and new trends 10.2.6 Light obscuration 10.2.6.1 Underlying principle and readout parameters 10.2.6.2 Application of LO during biopharmaceutical development 10.2.6.3 Considerations for LO measurements 10.2.6.4 Instruments and new trends 10.2.7 Flow imaging microscopy 10.2.7.1 Underlying principle and readout parameters 10.2.7.2 Application of FIM during biopharmaceutical development 10.2.7.3 Considerations for FIM measurements 10.2.7.4 Instruments and new trends 10.2.8 Visual inspection 10.2.8.1 Underlying principle and readout parameters 10.2.8.2 Application of visual inspection during biopharmaceutical development 10.2.8.3 Considerations for visual inspection measurements 10.2.8.4 Instruments and new trends 10.2.9 Brief overview of other methods 10.2.9.1 Flow cytometry 10.2.9.2 Microscopic techniques 10.2.9.3 Taylor dispersion analysis 10.3 General recommendations and pitfalls for particle analysis 10.4 Outlook and conclusions References 11 - Differential scanning calorimetry in the biopharmaceutical sciences 11.1 Background 11.2 DSC instruments 11.2.1 Solution DSC instruments—microcalorimeters 11.2.2 DSC instruments for solids 11.3 Practical considerations for DSC use 11.3.1 Solution DSC operational considerations 11.3.2 Sample handling 11.3.3 Resolution/sensitivity 11.3.4 Assessing the performance and reliability 11.3.5 Maintenance 11.3.6 Solid-DSC operational considerations 11.4 Data analysis 11.4.1 Solution DSC data analysis 11.4.2 Solid-DSC data analysis 11.5 Applications of solution DSC in biopharmaceutical Discovery and Development 11.5.1 Antibody therapeutics 11.5.2 Non-antibody protein therapeutics 11.5.3 Biosimilars 11.6 Applications of solid-sample DSC in biopharmaceutical discovery and development 11.7 Conclusions Acknowledgements References 12 - Biophysical mass spectrometry for biopharmaceutical process development: focus on hydrogen/deuterium exchange 12.1 Introduction 12.1.1 Protein primary structure 12.1.2 Higher-order protein structure 12.1.3 Native mass spectrometry 12.1.4 Ion mobility mass spectrometry (IM-MS) 12.1.5 Covalent labeling methods 12.1.6 Hydrogen/deuterium exchange 12.2 Synopsis of the technique 12.3 Mechanism of exchange 12.4 Advances in the technique 12.4.1 Deuteration 12.4.2 Quench/digestion 12.4.3 Separation 12.4.4 Mass spectrometry 12.4.4.1 Ionization technique 12.4.4.2 Mass analyzer 12.4.4.3 Tandem mass spectrometry and hydrogen scrambling 12.4.4.4 Stable isotope labeling 12.4.5 Automation 12.4.5.1 Automated data acquisition 12.4.5.2 Automated data reduction 12.5 Commercialization 12.6 Applications in the biopharmaceutical industry 12.6.1 HDX in molecular discovery 12.6.1.1 Structure-function relationship: establishing a mechanism of action 12.6.1.2 Epitope mapping, protein-protein and protein-ligand interactions 12.6.2 HDX in process development 12.6.2.1 Comparability and biosimilarity studies 12.6.2.2 Formulation development 12.6.2.3 Characterization of drug variant forms and process-related modifications 12.6.3 Characterization of emerging therapeutic entities 12.6.4 Sensitivity to structural or conformational variants 12.6.5 HDX-MS reproducibility, robustness, and best practices 12.7 Future perspective Acknowledgements References 13 - One- and two-dimensional NMR techniques∗ 13.1 Introduction 13.2 Physical basis of the technique 13.2.1 Basic theory and fundamental principles 13.2.1.1 Measurement of the NMR signal 13.2.1.1.1 Hardware required for the application: magnet size, probe design 13.2.1.1.1.1 The NMR spectrometer 13.2.1.1.1.2 Factors influencing sensitivity 13.2.1.1.1.3 Magnet 13.2.1.1.1.4 Probe design 13.2.1.1.1.5 Sample concentration 13.2.1.1.2 The chemical shift and the ppm scale 13.2.1.2 Decay of the NMR signal: relaxation influences both sensitivity and resolution 13.2.1.2.1 Two relaxation processes (T1 and T2), a Primer 13.2.1.2.2 Molecular size is the limit: fat molecules produce fat signals 13.2.1.3 NMR is a selective technique 13.2.1.3.1 Solvent suppression 13.2.1.3.2 Heteronuclear NMR techniques (link with selectivity by using isotope filtering) 13.2.1.3.3 Multi-dimensional NMR: example with 2D-1H-15N-HSQC 13.2.1.3.4 Faster data collection 13.2.1.3.4.1 The SOFAST-HMQC experiment 13.2.1.3.4.2 Non-uniform sampling (NUS) 13.2.1.3.5 Measuring diffusion of biomolecules: diffusion ordered spectroscopy (DOSY) 13.3 The appropriate technique for a particular problem 13.3.1 One-dimensional NMR: structure of small peptides and polysaccharides 13.3.2 Two-dimensional homonuclear versus heteronuclear 13.3.3 Pushing the molecular weight limit: 2D-NMR of monoclonal antibodies 13.3.4 Resolution of technique: magnet versus dimensionality 13.3.5 Some practical aspects on quantitative NMR 13.4 Method requirements and performance 13.4.1 NMR samples from biologic products 13.4.1.1 Sample preparation 13.4.1.2 Biologics are formulated: the challenge of excipients in NMR spectra 13.4.1.3 Albumin excipients 13.4.1.4 Filtering excipients and higher molecular weight signals using diffusion 13.4.2 Practicality and ease of use of the method: what calibrations are needed? 13.4.3 Amount of time required to make measurement (automation/sample throughput/amount of sample required/sample concentration range) 13.4.4 Assessing and maintaining instrument's performance 13.4.5 Need for internal referencing and controls 13.4.6 Sensitivity, reproducibility, and accuracy 13.4.7 Sample handling needs, issues and types of analytes 13.4.8 Sources of interference, limitations, errors and other sources of problems and how to potentially handle them (what samples ... 13.4.9 Level of technical training needed 13.5 Data processing (procedures) 13.5.1 Computer software: instrument vendors versus third party - pro and cons 13.5.2 Need for external processing and data evaluation systems (stand-alone or third party) 13.5.3 Statistical analysis procedures to assess level of uncertainty in information, especially in terms of assessing differences ... 13.6 Role in research versus process development 13.6.1 Role in research 13.6.2 Role in process development 13.6.3 Biosimilars 13.7 Technology update: recent and future advances and unique applications References Further reading 14 - Chromatography (other than size-exclusion chromatography) and electrophoresis 14.1 Introduction 14.2 Common features of concern when using chromatography and electrophoresis as biophysical characterization tools 14.2.1 Importance of maintaining a biopharmaceutical's native or native-like structure 14.2.2 Sample handling/processing 14.2.3 Nonspecific interactions 14.3 Chromatography 14.3.1 Chromatography particle 14.3.1.1 Chromatography particle (efficiency): size and structure 14.3.1.2 Chromatography particle (separations via selectivity): surface chemistry 14.3.1.2.1 Altering the selectivity of a given chromatography particle (surface chemistry) by changing the mobile phase 14.3.1.2.1.1 Changing mobile phase pH 14.3.1.2.1.2 Changing mobile phase chemical composition (salts, buffers and other additives) 14.4 Electrophoresis 14.4.1 Native electrophoresis in a gel format 14.4.2 Native electrophoresis in a capillary format: capillary zone electrophoresis (CZE) 14.4.3 Native isoelectric focusing electrophoresis (nIEF) 14.5 Some novel methods where chromatography or electrophoresis was used to help biophysical characterize protein biopharmaceuticals 14.5.1 Limited (or native) peptide mapping 14.5.2 Special form of native peptide mapping (analytical cascades of enzymes) 14.5.3 Self-interaction chromatography (SIC) and cross-interaction chromatography (CIC) 14.6 Characterizing the chromatographic and electrophoretic separations 14.7 The challenge that come with the ever-increasing higher resolution chromatography and electrophoresis when characterizing p ... 14.8 Summary References 15 - Challenges with the biophysical characterization of complex (multi-chain assembly, chemically modified, big and high concen ... 15.1 Introduction 15.2 Complex biopharmaceuticals I: multi-polypeptide chain protein in vivo assembly 15.2.1 Complex biopharmaceuticals II: multi-polypeptide chain protein in vitro disassembly and higher assembly (aggregation) via i ... 15.3 Complex biopharmaceuticals III: novel multi-polypeptide chain protein assembly in vivo to form bispecific antibodies 15.4 Complex biopharmaceuticals IV: via in vitro chemical modifications or in vivo recombinant coupling 15.4.1 Improving pharmacokinetics (PK) 15.4.2 Converting a non-therapeutic mAb and a toxic pharmaceutical (drug) into a therapeutic antibody drug conjugate (ADC) by chem ... 15.4.3 Assessing the impact of in vitro and in vivo modifications on the HOS of biopharmaceutical 15.5 Complex biopharmaceuticals V: assembly of large multi-protein biopharmaceutical structures – viral vectors (in gene therapy) 15.6 Complex biopharmaceuticals VI: high concentration biopharmaceuticals (mAbs) 15.6.1 High concentration biopharmaceuticals: searching for high colloidal and conformational stability 15.6.2 High concentration mAbs: searching for biopharmaceutical drug candidates with high colloidal and conformational stability 15.6.3 High concentration mAbs: unique opportunities in using AC-SINS to find mAbs with good colloidal stability 15.6.4 High concentration biopharmaceuticals: more rigors assessment of colloidal and conformational stability 15.7 Summary References 16 - The rigor of statistical analysis in assessing biophysical data: A lifecycle approach 16.1 Introduction 16.2 Description of the lifecycle approach 16.3 Characteristics of biophysical procedures 16.3.1 Typical biophysical procedures 16.3.2 The argument for statistical rigor 16.4 Statistical approaches in stage 1 of the lifecycle approach 16.4.1 Definition of procedure performance criterion (PPC) 16.4.1.1 Example 1: FUV-CD 16.4.1.2 Example 2: AUC 16.4.2 Determination of factors to include in the PPQ experiment 16.4.2.1 Example 1: FUV-CD 16.4.2.2 Example 2: SV-AUC 16.4.3 Sample size determination for the PPQ experiment (statistical power calculations) 16.4.3.1 Example 1: FUV-CD 16.4.3.2 Example 2: SV-AUC 16.5 Statistical approaches in stage 2 of the lifecycle approach 16.5.1 Example 1: qualification of FUV-CD 16.5.2 Example 2: qualification of AUC 16.6 Statistical assessment in stage 3 of the lifecycle approach 16.6.1 Procedure suitability test for FUV-CD 16.6.2 Procedure suitability test for AUC 16.6.3 Control charting reference material 16.7 Considerations for successful implementation of a lifecycle approach: management of biophysical procedures 16.8 Summary References 17 - Developability in biophysical characterization 17.1 Introduction 17.2 Computational structural methods and their use in lead candidate selection and in engineering 17.2.1 Computational structural methods: aggregation 17.2.2 Computational structural methods: viscosity 17.2.3 Computational structural methods: other properties 17.2.4 Computational structural methods: in silico approaches 17.3 Formulation considerations for developability screening 17.4 Critical quality attribute based testing in developability 17.4.1 Protein aggregation (including submicron and subvisible particles) 17.4.1.1 Size exclusion high/ultra performance liquid chromatography (SE-H/UPLC) 17.4.1.1.1 SE-H/UPLC with multi-angle light scattering (MALS) 17.4.1.2 Dynamic light scattering (DLS) 17.4.1.3 Submicron and subvisible particles 17.4.2 Post-translation modifications (PTMs) 17.4.2.1 Aspartic acid isomerization 17.4.2.2 Asparagine deamidation 17.4.2.3 Methionine oxidation 17.4.2.4 Tryptophan oxidation 17.4.2.5 Fragmentation 17.5 Colloidal stability: predicting protein solution behavior 17.5.1 Colloidal stability: second virial coefficient 17.5.2 Colloidal stability: diffusion interaction parameter (kD) 17.5.3 Colloidal stability: affinity capture self-interaction nanoparticle spectroscopy (AC-SINS) 17.5.4 Colloidal stability: zeta (ζ-) potential 17.5.5 Colloidal stability: summary 17.6 Viscosity and various mature and emerging viscosity technologies 17.6.1 Predictive and direct/indirect viscosity measurements 17.6.1.1 Viscosity: VROC® initium 17.6.1.2 Viscosity: viscosizer TD 17.6.1.3 Viscosity: via dynamic light scattering 17.7 Conformational stability 17.7.1 Conformational stability: differential scanning calorimetry (DSC) 17.7.2 Conformational stability: differential scanning fluorimetry (DSF) 17.7.3 Conformational stability: isothermal chemical denaturation (ICD) 17.8 Conclusion and future of aspects of developability References 18 - Technical decision making using biophysical data 18.1 Introduction 18.2 Current perspectives and recent trends 18.3 Future direction 18.3.1 Continued growth in the depth and breadth of biophysical techniques 18.3.2 Toward a risk-based and right-sized biophysical strategy 18.3.3 Biophysics – one piece of a larger puzzle 18.4 Conclusion Acknowledgements References 19 - Biophysical characterization: an integral part of the “totality of the evidence” concept 19.1 Biopharmaceutical development 19.1.1 Drug approval: “one-size-fits-all” versus “case-by-case” 19.2 An introduction to the “totality of the evidence” and its more global meaning in developing biopharmaceuticals 19.3 Biophysical characterization in developing protein biopharmaceuticals 19.4 Building a Biopharmaceutical's biophysical fingerprint 19.4.1 Building a Biopharmaceutical's biophysical fingerprint (part I): using the standard (basic or core) biophysical characteriz ... 19.4.2 Building a Biopharmaceutical's biophysical fingerprint (part II): via biophysical properties 19.4.3 Building a Biopharmaceutical's biophysical fingerprint (part III): via advanced (high-resolution) biophysical tools 19.4.3.1 Getting access to advanced biophysical instruments/expertise 19.4.3.2 Better resolution is likely to reveal the presence of small differences: are these differences important? 19.5 Detecting small differences in biopharmaceuticals via biophysical characterization measurements 19.5.1 Difference—Outside the uncertainty level (precision or reproducibility) of a biophysical characterization measurement 19.5.2 Difference—Staying within the limits of allowable manufacturing variability - biopharmaceutical consistency and comparabili ... 19.6 Conclusion References Index A B C D E F G H I K L M N O P Q R S T U V W X Z