From spotting contaminating microbes in cell cultures to verifying nucleic acid sequences for mRNA therapeutics with single base pair resolution, capillary electrophoresis plays a versatile and critical support role in manufacturing advanced therapies. Capillary electrophoresis allows researchers to perform Sanger sequencing and fragment analysis to spot potentially dangerous errors early during therapeutic development.
Download this poster from Thermo Fisher Scientific to learn how to leverage this versatile tool for efficiently and safely generating therapeutic antibodies, mRNA vaccines and therapeutics, and cell and gene therapies.
The role of capillary electrophoresis in manufacturing advanced therapies
The art of simple
Capillary electrophoresis (CE) is a simple technique that enables accurate, efficient, and safe therapeutics. CE uses an electric charge to separate and migrate labeled nucleic acids through a polymer coated capillary. Unlike gel separation, CE offers superior sensitivity, faster separation, and higher throughput from smaller sample inputs. CE is a highly versatile tool that empowers both single molecule resolution for Sanger sequencing of mRNA vaccines and therapeutics and delivers information on biomolecule size and concentration for fragment analysis during cell and gene therapies.
Verifying cell type
Many therapeutics rely on cell lines for their production, such as Chinese hamster ovary (CHO) cells for antibodies or Henrietta Lacks (HeLa) cells for vaccines and cancer therapies. Cell lines can become contaminated with unrelated cells or misidentified, leading to inaccurate research and safety conclusions. Fragment analysis using CE allows researchers to confirm cell lines based on established profiles (2).
Sanger sequencing
Since sequencing the human genome in 2003, Sanger sequencing has remained the gold standard for fast, simple, and cost-effective DNA sequencing. Sanger sequencing by CE reads up to 1,000 bases per reaction at single base pair resolution with 99 percent accuracy and without complicated next generation sequencing protocols or data analysis.
Matching cells and donors
Induced pluripotent stem cells (iPSCs), differentiated cells taken from patients and reprogrammed into a stem celllike fate, promise precision cell therapy and drug discovery. CE enables researchers to molecularly fingerprint alleles at different genomic loci and efficiently match iPSCs after ex vivo manipulation to their donors, optimizing therapeutic efficacy and minimizing immunogenicity (3).
Fragment analysis
Fragment analysis uses a simple CE workflow to profile nucleic acid fragments based on their sizes and migration properties. CE accurately sizes fragments that differ by only one base pair, facilitating high resolution fragment analysis. Research can also easily automate and multiplex fragment analysis protocols using CE, enabling cost effective and fast turnaround times.
Identifying viral capsids
Adeno-associated viruses (AAVs) serve as delivery vehicles for therapeutic genes. Delivering empty AAVs can increase gene therapy immunogenicity. Recent approaches using fragment analysis to detect fluorescently labeled AAV viral proteins allow researchers to determine the number of full versus empty AAVs, as well as identify different AAV serotypes, which could have implications for gene therapy tropism (4).
A simple solution to avoiding the dangerous cost of getting it wrong
Numerous early steps go into producing a safe and effective therapeutic. Despite researchers’ diligence, errors can creep into this process from missed point mutations to hidden microbes that can cause a therapeutic to work inadequately or have devastating immunological side effects. CE’s role in Sanger sequencing and fragment analysis places it at the center of various early, but critical quality control steps to ensure safe and effective therapeutics.
Spotting microbes
Microbes can infect cell cultures and cause chromosomal aberrations in cells needed to produce mRNA vaccines and therapeutics, monoclonal antibodies, and cell and gene therapies. Microbe-induced changes alter cell functions, including nucleic acid synthesis and membrane antigenicity (5), that affect research results and pose significant health risks to patients. Sanger sequencing using CE identifies microbial contamination.
Confirming nucleic acid sequences
During mRNA vaccine and therapeutic production, researchers manipulate DNA and RNA intermediates using plasmids, which may cause unwanted mutations. Using Sanger sequencing during plasmid production and mRNA identity testing provides a simple, scalable, and reproducible approach for confirming the sequence accuracy of mRNA therapeutics (6).
References
1. What are the applications of capillary electrophoresis? Thermo Fisher Scientific https://www.thermofisher.com/us/en/home/lifescience/sequencing/sequencing-learning-center/capillary-electrophoresis-information/capillary-electrophoresis-applications.html
2. Almeida, J.L. & Korch C.T. Authentication of human and mouse cell lines by short tandem repeat (STR) DNA genotype analysis. Assay Guidance Manual [Internet] (2023).
3. Matching identities of iPSCs and donors using CLA IdentiFiler STR profiling kits. Thermo Fisher Scientific http://assets.thermofisher.com/TFS-Assets/GSD/Reference-Materials/stem-cell-line-authentication-whitepaper.pdf
4. Lock, S.J., Hendriks, K., & Berlet, J. The use of capillary electrophoresis in gene therapy. Cytotherapy 22, S152 (2020).
5. Nikfarjam, L. & Farzaneh, P. Prevention and detection of mycoplasma contamination in cell culture. Cell Journal 13, 203-212 (2012).
6. mRNA manufacturing QC using Sanger sequencing. Thermo Fisher Scientific https://www.thermofisher.com/us/en/home/life-science/sequencing/sanger-sequencing/applications/mrna-manufacturing-qc.html