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What is cell and gene therapy?

Advances in genetics and bioengineering have enabled the development of gene therapy and with it, the unprecedented potential to treat diseases. Gene therapy involves the addition or modification of a defective gene in a patient's cells with a healthy version of that gene to treat, prevent or cure disease.

 

Gene delivery systems are required to deliver gene therapies to target cells and currently the most common delivery system is viral vector delivery utilizing adeno-associated viruses (AAVs).

 

Technologies for manufacturing and analysis of AAV-based gene therapy products are evolving and remain to be fully optimized.

 

Producing safe, effective, and reproducible AAV-based gene therapies can create many additional analytical challenges.

Choosing the right sample vial for analysis of AAV to prevent sample loss

Gene Therapy Analytical Solutions Guide

Cell and Gene Therapy Solutions

Gene Therapy Analysis

Cell and gene therapies workflows.


AAV identity confirmation

AVVs must be well-characterized to understand the relationship between product quality attributes, manufacturing processes, patient safety and efficacy. Several approaches are often used collectively to comprehensively characterize AAV-based gene therapies.

 

Capsid protein characterization

 

It is important to understand the capsid identity because each AAV serotype has unique capsid proteins that transfer the genetic material to specific cells or organs. Depending on the therapeutic target area, the correct AAV serotype is chosen, and identity and purity are monitored. All AAV capsids consist of three proteins (VP1, VP2, and VP3) that share high-sequence homology, and ensuring the identity and purity of these proteins are critical to viral infectivity and viral transfer. Given the importance of the serotype and the capsid protein composition, there exists a need for methods to identify and monitor the capsid and the capsid proteins through gene therapy development and manufacturing.

AAV capsid protein characterization workflow.

A Detailed Look at Antibodies, Plasma Proteins, Viruses and Gene-Delivery Vehicles by High-Resolution Native Mass Spectrometry

Collaborator: Professor Albert J.R. Heck. Utrecht University

Emerging LC-MS Techniques to Characterize AAV at the Subunit and Intact Levels

Collaborator: Regeneron

Demonstrated Benefits of Low-flow Analytical Methods for Biopharmaceutical Applications

Collaborator: NIBRT

Primary sequence and PTM characterization

 

In addition to identifying AAV serotypes and associated capsid proteins (and resulting ratios), one must characterize capsid proteins at the primary sequence level including analysis of post translational modifications (PTMs). This sequence level characterization is essential ensure product quality and consistency.

AAV Primary sequence and PTM characterization workflow.

An automated high-throughput workflow for peptide mapping to monitor post-translational modifications (PTMs) of monoclonal antibodies

Collaborator: NIBRT

Investigating process-related post-translational modifications in NISTmAb RM 8671 using high-throughput peptide mapping analysis

Collaborator: NIBRT

Peptide mapping of challenging monoclonal antibodies

Collaborator: Symphogen

Characterizing Therapeutic Proteins for Drug Discovery and Development

Collaborator: LifeArc

See more resources

Intact capsid mass analysis

 

One important quality attribute of AAV products that requires monitoring is the amount of residual empty capsids following downstream processing. Traditionally, empty and full particles are quantified via analytical ultracentrifugation as well as anion exchange chromatography using ultraviolet or fluorescence detection. A native mass spectrometry-based approach to assess the ratio of empty to full AAV-capsids without the need for excessive sample preparation has recently shown great promise.

Analytical solutions for intact mass analysis

AAV intact capsid mass analysis workflow.

A Native Mass Spectrometry-Based Assay for Rapid Assessment of the Empty: Full Capsid Ratio in Adeno-Associated Virus Gene Therapy Products

Collaborator: NIBRT

Quantitative analysis of genome packaging in adeno-associated viruses using native MS

Collaborator: NIBRT

Viral titre analysis of AAV viral capsids

 

Among others, one important step post-upstream production of AAVs is to accurately determine the titre of the viral capsids. Size exclusion chromatography with florescence detection can be utilized to determine viral capsid titre following upstream purification.

Analytical solutions viral titre of AAV viral capsids

Viral titre analysis workflow for AAV viral capsids.

Size exclusion chromatography with fluorescence detection to determine AAV titers following downstream processing

Collaborator: NIBRT

 


AAV impurity analysis

During the gene therapy development process, impurities and degradation products can be formed during the manufacturing process or storage of the drug. Regulatory guidance requires that studies be conducted to identify the impurities present and to characterize their structure.

 

Identification and quantification of both process and product related impurities is necessary. Product-related impurities relate to molecular variants with properties different from those of the desired product formed during manufacture and storage. Process related impurities are related to the manufacturing process and may include for instance cell substrates.

 

Host cell protein analysis

 

Host cell proteins (HCPs) are low-level, process-related protein impurities in drug products derived from the host organism during biotherapeutic manufacturing. During expression of a biotherapeutic drug, host cell systems can express many endogenous proteins.

 

Purification of the drug from these HCP contaminants can be challenging, with low-level contamination remaining after purification. Host cell proteins (HCPs) accompanied with biotherapeutics can significantly affect drug efficacy and cause immunogenicity. The detection and quantification of residual HCPs as potential process-related impurities is critical for biopharmaceutical companies in accordance with regulatory agency guidelines (ICH Q6B).

AAV host cell protein analysis workflow.

Demonstrated Benefits of Low-flow Analytical Methods for Biopharmaceutical Applications

Collaborator: NIBRT

 

Empty/full capsid analysis

 

Empty capsids, which do not contain the gene therapy of interest, and partial capsids, those containing only a fragment of the gene of interest, are by-products of the AAV production and can impact product safety and efficacy. The amount of full, partial, and empty capsids, therefore, needs to be characterized and monitored through process development.

 

A Native Mass Spectrometry-Based Assay for Rapid Assessment of the Empty:Full Capsid Ratio in Adeno-Associated Virus Gene Therapy Products

Collaborator: NIBRT

Quantitative analysis of genome packaging in adeno-associated viruses using native MS

Collaborator: NIBRT

Aggregate analysis

 

Due to the immunogenicity risks of aggregates, this is a critical quality attribute that must be fully defined and monitored. Aggregates are formed during production and storage. Size exclusion chromatography (SEC) methods provide an inexpensive approach to monitoring aggregation in a high-throughput manner.

 


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