Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics

Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics
Copyright
Contributors
Author Biographies
Introduction
1. LC-MS characterization of antibody-based therapeutics: recent highlights and future prospects
	Section 1 – LC-MS technologies frequently used for the characterization of antibody-based therapeutics
		Analysis of intact antibodies
		Middle-up analysis
		Bottom-up analysis
		Higher order structure analysis by mass spectrometry
		Host cell protein analysis
	Section 2 – LC-MS characterization of antibody attributes
		Confirmation of primary sequence and detection of cleavages
		Cysteine modifications
		N-linked glycosylation
		O-linked glycosylation
		Glycation
		Deamidation and aspartic acid isomerization
		Oxidation
		Drug conjugates
		Other modifications of antibodies and antibody fusion proteins
	Section 3 – Enabling technologies and future directions for LC-MS-based antibody characterization
		New reference materials
		Enabling enzymes
		Top-down and middle-down analysis
		Native mass spectrometry
		Ion mobility mass spectrometry
		Alternative chromatographies for antibody and ADC analysis
		Multi-Attribute Method
	Conclusion
	References
2. Engineering of Protein A for improved purification of antibodies and Fc-fused proteins
	Antibody purification
		Antibody binding proteins
			Staphylococcal Protein A
	Protein A-based purification
	Engineering of Protein A domains for enhanced alkaline stability
	Milder elution conditions in Protein A-based chromatography
		Elution buffer additives for milder elution
		Structural engineering for improved elution
		Loop engineering
	Improving the binding capacity of Protein A resins
		Base matrices and ligand density
		Ligand multimerization
			Optimization of the linker regions
		Ligand coupling
	Summary
	References
3. High fidelity affinity purification of Fc-fusion molecules from product related impurities
	Introduction
	How affinity resins are discovered, developed and manufactured
	Case study 1: Fc-fusion protein #1
	Case study 2: Fc-fusion protein #2
	Case study 3: A general approach to purification of bispecific antibodies
	Case study 4: heterodimer specific resins
	Summary
	References
4. Recent advances in continuous downstream processing of antibodies and related products
	Introduction
	Introduction: drivers for change in biopharm manufacturing – changing landscape
		Biosimilars
		Multiple mAbs for the same indication
		Intensified upstream process and the downstream bottleneck
		Small molecule success and regulatory drive
		Pros and cons of continuous processing
	Key enabling technologies for continuous processing
		Acoustic wave separation
			Multicolumn chromatography
			Low pH virus inactivation
			Continuous stirred tank reactors
			Cyclical flow reactors
			Column based strategies
			Alternatives to low pH virus inactivation
	Single-pass tangential flow filtration
		Single-pass tangential flow filtration (SPTFF)
		SPTFF for in line diafiltration (ILDF)
			Updates from early adopters
			Alvotech
			Amgen
			BiosanaPharma
			Boehringer ingelheim (BI) and pfizer
			Enzene
			Genzyme
			Just Biotherapeutics
			Merck
			Novartis
	Conclusion: tipping point
	References
5. Recent advances in antibody-based monolith chromatography for therapeutic applications
	Introduction
	State of the art of antibody-based monolith chromatography
		Generalities and fundamentals
		Advantages and limitations
	Recent advances in purification of antibodies
	Recent advances in immobilization of antibodies
	Therapeutic applications
		Diagnosis and analysis
		Treatment
	Future trends and concluding remarks
	References
6. Recent advances in harvest clarification for antibodies and related products
	Introduction
	Advances in mechanical separation methods
	Encapsulated depth filters
	Non-woven depth filters
	Synthetic depth filters
	Body feed
	Pretreatment technologies
	Cell retention devices
	Analytical technologies for clarification
	References
7. Recent advances in ultrafiltration and virus filtration for production of antibodies and related biotherapeutics
	Introduction
	Virus filtration
		Introduction
		Recent regulatory trends
			Virus filter classification and use
			Modular virus clearance validation
			Quality by design
			Virus filter clearance validation using only small viruses
		Relevant process trends
			Increased mAb concentration
			Connected and continuous processing
			Virus clearance validation for connected and continuous processes
			Process pauses
			Filtration as an upstream viral barrier
		Filtration technology development
			Membranes and filtration devices
			Adsorptive prefiltration
			Virus retention mechanisms
	Ultrafiltration
		High concentration formulation
			High solution viscosity
			Low final batch volume
		Excipient offsets
		Single-pass tangential flow filtration
			Single-pass TFF operation
			Applications for single-pass TFF
			SPTFF system considerations
		Single-use tangential flow filtration
	References
8. A roadmap for IgG-like bispecific antibody purification
	Introduction
	Asymmetric bsAb
		Homodimer
			bsAb constructed using the KiH technology
			bsAb with electrostatic interaction driven heterodimerization
			bsAb based on WuXiBody platform
			bsAb with pI engineered HC
			bsAb with Protein A-binding abolished HC
			Common HC bsAb - κλ-body
		Single-chain variable fragment (scFv)-Fc
		Half antibody
	Symmetric bsAb
		Protein A chromatography
		Hydrophobic interaction chromatography (HIC)
		Capto MMC ImpRes and Capto adhere chromatographies
		HA chromatography
		CEX chromatography
	Purification roadmap
	Concluding remarks
	Acknowledgment
	References
9. High-throughput, parallelized and automated protein purification for therapeutic antibody development
	Introduction
	Small-scale equipment and strategies
		Liquid handling platforms
		Resin-filled purification tips
		Magnetic beads
		Plate-based membrane chemistries
		Batch purification in plates
		Robo columns
		Buffer exchange and protein concentration
	Mid-scale purification strategies
		Batch methods
		Automation options
		Protein Maker
		Modified FPLC and HPLC systems
		Magnetic resin based separations
		Membrane-based and monolith devices
	Applications to HT process development
	Non-chromatographic HT purification approaches
		Batch crystallization
		Precipitation
		Aqueous two-phase systems
	Summary and conclusions
	References
Index
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