Within cell and gene therapy lies a diverse range of therapeutic approaches all aimed at treating or curing disease by either blocking, altering or augmenting the expression of specific genes.
Gene Therapy
Gene therapy is the in vivo delivery of a nucleic acid sequence where multiple modalities exist, but in general, they can be grouped into three categories: 1) gene silencing/modulation 2) gene insertion/replacement and 3) gene editing. Delivery is typically addressed via encapsulation in viral vectors (ie. adeno-associated virus (AAV)), or lipid nanoparticles (LNPs), which become an integral part of the drug product and must be fully characterized as well.
Cell Therapy
Cell therapy is when the nucleic acid is delivered into cells ex vivo and these engineered cells are subsequently delivered into a patient. Both autologous and allogeneic cell therapies are being developed today.
At their core, cell and gene therapies are comprised of nucleic acids, proteins and lipids that need to be fully characterized and for which precise chemistry, manufacturing and control (CMC) processes must be established to ensure their safety and efficacy. Waters offers a broad set of tools, solutions and expertise in biomolecular separations, mass analysis and biophysical characterization to support the development and commercialization of these new and exciting modalities.
Nucleic AcidsNucleic acid-based therapeutics, such as antisense oligonucleotides and RNAi therapies, routinely incorporate modified nucleotides for greater stability and are often conjugated to other molecules to effect targeted delivery. Being able to separate these molecules from a multitude of synthesis related impurities is critically important for their characterization and CMC testing, impurities analysis, and QC release. Additionally, it is critically important to confirm the nucleotide se-quence including the location of each modification within the sequence (aka: modification mapping). To this end, Waters offers unique high-performance column chemistries and fit-for-purpose LC-MS workflows that deliver unparalleled separations performance for oligonucleotides and support everything from characterization and sequence confirmation to GMP validated QC release. Featured Technology: BEH & ACQUITY PREMIER column technology, ACQUITY UPLC systems, BioAccord LC-MS system, XEVO G2-XS QTof, Vion & SYNAPT XS MS platforms, MassPREP standard, waters_connect, MassLynx, ProMass, Spectrum Tools |
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One way to deliver nucleic acid therapeutics to cells is to encapsulate them in lipid nanoparticles (LNPs). The size, composition, charge, and surface chemistry of LPNs are varied to improve delivery selectivity. ITC and DSC can both be used to interrogate the higher-order complexes formation and structure. RP chromatography is added to provide detail and GC-MS facilitates ID and detection of impurities of the raw materials.
Featured Technology: Charged Surface Hybrid™ C18 column chemistry, ACQUITY UPLC, Xevo TQ-GC, SFC, Nano DSC, Nano or Affinity ITC
Viral VectorsThe presence of the desired transgene encapsidated within a viral vector is a critical quality attribute (CQA) and because there is a charge difference between the two species, quantification and separation of empty/full viral vectors can be completed using anion exchange chromatography. Additional elements to monitor that impact transfection efficiency, selectivity, and immunogenicity include the protein subunit ratio, post-translational modifications (PTMs), and size variants. The first two are best addressed by peptide mapping and the latter can be assessed using size exclusion chromatography. Featured Technology: BioAccord LC-MS System, UNIFI, ACQUITY UPLC H-Class PLUS Bio System, ACQUITY UPLC with FLR Detector, RP C4 300Å or C18 300Å column, Protein Pak, XBridge Protein BEH450 SEC Columns, Empower Software with Auto Blend Plus Technology, Protein-Pak Hi Res Q Column |
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Bioprocess Attribute MonitoringMetabolite monitoring is a useful process in the analysis of cell culture media whether it is for a viral vector product or cell therapy propagation of either autologous or allogeneic cells. In untargeted mapping, a researcher is given freedom to discover and after the best predictors are discovered, an automated targeted validation method with Progenesis can increase efficiency. Featured Technology: Progenesis QI, UNIFI, MassLynx, SFC, HRMS, HILIC, Triple Quad MS/MS, Patrol, AccuTag |
Genomic and Cellular Biology Automation and PreparationScalable, consistent identification and quantitation of nucleic acids requires both accuracy and traceability built into an automated workflow. These requirements can be achieved using the highly versatile Andrew+ pipetting robot and Pipette+ smart pipetting system, benefitting from the design and execution of protocols for RT-PCR, Next Generation Sequencing, clonal isolation, and plasmid purification. Moving into to larger complexes, cell preparations for flow cytometry and staining can also be prepared using the same automation system. Featured Technology: Andrew+, OneLab, BeadTender, Magnet+ www.AndrewAlliance.com
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Short Nucleic Acids (ASO, siRNA, gRNA)
and Long Nucleic Acids (mRNA, pDNA)
Purity, Aggregation
•Arc or Alliance HPLC: RP, IEX • UPLC: IEX, HILIC, Affinity • LC: SFC •SEC
Impurities
• LC/MS Quad • LC/MS ToF or ion trap, • Arc or Alliance HPLC
Titer
• Arc or Alliance HPLC: RP or IEX; • UPLC: IEX, HILIC, Affinity • SEC
Separation pDNA from other forms
• UPLC • SEC
Length, Sequence
• Arc or Alliance HPLC: RP, IEX • UPLC/MS: Tof, ion trap
Modifications, Post-Transcription Changes
• LC/MS: Tof, ion trap
Complex detection and binding
• LC/MS • Affinity ITC • Nano DSC
Conformational Stability
• Nano DSC
Raw materials, Drug Product, Drug Substance
Purity, Drug Substance
• GC-MS • Nano DSC • DSC
Impurities: Raw Materials
• GC-MS • GPC
Complex Formation and Stability
• Affinity ITC • Nano DSC
ID: Lipid Separation
• UPLC:RP
Viral Vectors, CRISPR-Cas
Empty vs Full
• UPLC: IEX, FLR detector
Impurities, Host Cell Proteins
• UPLC-MS • SEC
Capsid Titer
• SEC, FLR or UV Detector
Capsid Protein Ratio, Variants
• Intact protein analysis • UPLC, FLR or UV detector • UPLC-MS
Capsid ID, PTM, Sequence Variants, N-glycan
• Intact protein analysis • UPLC, FLR or UV detector • UPLC-MS
Conformational Stability
• HDX-MS • Nano DSC
Nucleoprotein Complex (CRISPR)
• UPLC • Nano DSC
Metabolite Monitoring and ID
Cell Propagation and Production
• UPLC HRMS • UPLC: HILIC, RP, SFC
ID Predictors of Cell Health
• UPLC HRMS • UPLC: HILIC, RP, SFC
Targeted Analysis
• Triple Quad MS/MS • UPLC HRMS • UPLC: HILIC, RP, SFC • SEC
Map Biomarkers and Pathway
• Progenesis Qi
Front-End Workflow: Flexibility, Traceability and Accuracy
RT qPCR
• Andrew+ • OneLab • Promega GoTaq • Single-Step SYPR® Green
DNA Plasmid Prep
• Andrew+ • Pipette+ • Macherey-Nagel •Nucelobond Midiprep • Nucelospin Viral RNA
Next Generation Sequencing Libraries
• Andrew+ • KAPA DNA Library • DNA Sequencing Library
Flow Cytometry Preparation
• Andrew+
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