Gene therapy

Viruses and their derivatives have many applications in biomedicine, bio- and nanotechnology. In gene therapy, viruses are used as vectors to deliver modified genes into target cells, to treat or prevent a disease by: 

  • Replacing a mutated gene that causes disease with a healthy copy of the gene
  • Inactivating or ‘knocking out’ a mutated gene that is functioning improperly
  • Introducing a new gene into the body to help fight a disease or treat a syndrome

Adenoviruses, retroviruses, lentiviruses, adeno-associated viruses (AAVs) and other animal viruses have been utilized for the development of gene therapies over several decades. In recent years, this application area has seen significant growth, largely due to developments in recombinant AAV vector engineering and production for in vivo gene therapies.

In order to develop efficacious gene therapy vectors which are produced through controlled and economical manufacturing processes, multiple challenges must be addressed.  These range from capsid design through the identification of optimal process and formulation conditions, to comprehensive quality control of drug substances and drug products. 

Application of Malvern Panalytical solutions across the in vivo gene therapy development workflow

From capsid design, through the optimization of downstream process conditions, to formulation and stability tests and the extended characterization of drug substances and drug products, technologies such as Dynamic Light Scattering (DLS), Electrophoretic Light Scattering (ELS)Multi-Angle Dynamic Light Scattering (MADLS), Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS), Nanoparticle Tracking Analysis (NTA), Isothermal Titration Calorimetry (ITC) and Differential Scanning Calorimetry (DSC) are used to inform scientists on the key analytical and quality attributes of viral vectors, enabling characterization, comparison and optimization of:

  • Capsid size (DLS, SEC, NTA)
  • Capsid titer or particle count (MADLS, SEC, NTA)
  • Percentage of genome-containing virus particles / % full analysis (SEC
  • Aggregate formation (DLS, MADLS, SEC, NTA)
  • Fragmentation (SEC)
  • Thermal stability (DLS, DSC)
  • Higher-order structure analysis (DSC)
  • Serotype identification (DSC)
  • Capsid uncoating and genome ejection (DLS and DSC)
  • Binding to receptor (ITC)
  • Charge (ELS)

DLS, MADLS, SEC-MALS, NTA, ITC, and DSC are label-free biophysical techniques which require minimal assay development and can be readily applied at all stages, strengthening the analytical workflow for gene therapy development.

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Research and early development: viral capsid design

Although the discovery process for gene therapy is shorter than that typically seen in traditional drug discovery, the high degree of product complexity introduces additional challenges which must be addressed early-on to assure the delivery of safe and efficacious products. Amongst these challenges are:

  • Selection of a viral capsid based on optimal properties and function
  • Rational protein engineering to improve and modify the properties and functionalities of the original viral capsid

The solutions in both cases are based upon a comprehensive set of physicochemical, biochemical, and biological data which informs on the performance of the viral vector and feeds back on the selection process. 

At this stage, extensive biophysical characterization of engineered capsids and viral vectors using DLS, MADLS, SEC-MALS, ITC, and DSC supports the reliable evaluation of important quality metrics and interpretation of the results of biochemical and biological assays, via measurements of capsid size and titer, aggregate formation, % full measurement, receptor binding, thermal stability and capsid uncoating propensity.

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Zetasizer Ultra

World’s most advanced light scattering system
Zetasizer Ultra

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The world’s most advanced multi-detector GPC/SEC system

Gene therapy process development

The gene therapy production process must meet strict regulatory requirements and other internal expectations for quality, timelines and costs. Fit-for-purpose solutions are needed to support and strengthen the analytical workflow and answer challenges concerning: 

  • High degree of product complexity
  • Diversity of viral vectors for gene delivery in design and development 
  • Suboptimal downstream processing with lengthy analytical assays suffering from significant variability

Throughout the downstream purification process, multiple assays are performed to determine the key analytical attributes that determine yield and report on Critical Quality Attributes (CQAs) such as viral vector purity, potency, stability and safety.  These parameters are typically, but not limited to, the following:  

  • Capsid titer or particle count
  • Genome count
  • Percentage of genome-containing virus particles or % full analysis
  • Serotype characterization
  • Aggregate formation 
  • Contamination by unwanted host-cell proteins and nucleotides 

The first three parameters (capsid titer, genome count, % full analysis) are commonly measured using two or more of the following assays:  qPCR, ddPCR,  ELISA, AUC, HPLC-AEX, and/or TEM.  Each method has intrinsic strengths and weaknesses relating to the parameter measured, throughput, speed, accuracy, and sample volume requirements. 

Gene therapy: Characterize, compare, optimize

In process development for viral vectors such as AAVs, the Zetasizer Ultra is well-suited as a complementary assay that can be utilized in existing analytical workflows and provides a rapid, label-free, non-destructive, low volume, and orthogonal measurement of total virus particle concentration, capsid titer, capsid size, charge, aggregate formation, thermal stability and capsid uncoating.

Accurate and precise size analysis is essential to the particle concentration measurement. The Zetasizer Ultra utilizes three scattering angles to provide a more precise, higher resolution measurement. In Multi-Angle Dynamic Light Scattering (MADLS), scattering information from the back, side and forward angles is collected and combined into a single higher resolution size distribution which provides more representative data.

Size exclusion chromatography (SEC) has long been used as a key tool to measure the molecular weight of macromolecules, proteins, viruses, polysaccharides and polymers. OMNISEC, a multi-detection SEC system, can provide data on several key analytical and quality attributes of AAVs, such as capsid and genome titer, and also % full – these are not accessible via UV detection only. These important parameters provide vital information on viral vector purity, potency and stability.

Differential Scanning Calorimetry (DSC) is a well-established tool in the characterization and development of virus-based products, including several commercial vaccines. In addition to multiple stability metrics for viral vectors, DSC provides a TM of capsid disintegration which is characteristic of a serotype ID, it maps thermal stability, fingerprints higher order structure, and can detect structural changes in response to stress, formulation or process condition changes.

Viral capsid stability and function are locked in a fine balance. Viral capsids must be stable enough to contain and protect the genome, bind to the host cell surface for cellular uptake and navigate the cellular milieu. However, a viral capsid must also offer enough conformational lability to release the genome at the replication site.

The mechanism of AAV vector uncoating remains poorly understood, but structural change appears to be required for capsid uncoating and genome release.  Viral vector propensity to uncoat is postulated to correlate with an important quality attribute - infectivity. DSC, in conjunction with Dynamic Light Scattering thermal ramps, can be used to assess the uncoating propensity of a viral capsid in response to buffer and stress conditions.

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Zetasizer Ultra

World’s most advanced light scattering system
Zetasizer Ultra


The world’s most advanced multi-detector GPC/SEC system

MicroCal DSC range

The "Gold Standard" for structural stability analysis of biotherapeutics, b...
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