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Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS
Published on: June 20, 2019
Analytical Characterization of Pneumococcal Vaccine Conjugates Using SEC-MALS Technique
Chloe Wright1, Gowri Chellappan1, Abigail Mydland1
1Bacterial Research and Development, Inventprise, Inc., Redmond, WA 98052, USA.
Insights
Size-exclusion chromatography-multi-angle light scattering (SEC-MALS) accurately characterizes pneumococcal conjugate vaccines. This method provides detailed insights into molecular attributes, aiding in the development of more effective vaccines.
Area of Science:
- Vaccinology
- Analytical Chemistry
- Biophysical Characterization
Background:
- Streptococcus pneumoniae infections cause severe illness, necessitating effective conjugate vaccines.
- Developing pneumococcal conjugate vaccines requires detailed characterization of polysaccharide and protein components.
- Existing analytical methods may not fully capture the complexity of conjugate vaccine structures.
Purpose of the Study:
- To demonstrate the utility of size-exclusion chromatography-multi-angle light scattering (SEC-MALS) for pneumococcal conjugate vaccine characterization.
- To evaluate SEC-MALS for assessing key attributes like molecular weight, composition, and conjugation efficiency.
- To showcase SEC-MALS as a tool for optimizing vaccine development and manufacturing.
Main Methods:
- Utilized SEC-MALS to analyze pneumococcal conjugate vaccines.
- Employed specific capsular polysaccharides (STs 1, 3, 5, 10A, 18C, 24F, 33F) conjugated to rCRM197 carrier protein.
- Validated the technique using established standards for accuracy and repeatability.
Main Results:
- SEC-MALS demonstrated high accuracy (>90%) and repeatability (<2% RSD) for molar mass measurements.
- The technique provided detailed information on conjugate molecular assembly and conformation.
- Compositional analysis enabled refinement of the conjugation process to achieve desired molar masses.
Conclusions:
- SEC-MALS is a valuable tool for in-depth biophysical characterization of conjugate vaccines.
- Accurate characterization is crucial for optimizing vaccine product attributes and ensuring manufacturing consistency.
- This technique supports the development of potent and reliable pneumococcal conjugate vaccines.
Background/Objectives:
Infection from Streptococcus pneumoniae can lead to serious complications, such as meningitis and pneumonia, in children under 2 years of age, older adults, and immunocompromised populations. Conjugate vaccines against the pathogen have been licensed for the prevention of invasive pneumococcal disease. Conjugate vaccine development is an involved process demanding extensive characterization of both the polysaccharide (PS) and protein (Pr) moieties in complex structures. One powerful tool in our analytical tool kit that can shed light on various analytical attributes of conjugate vaccines, such as molecular weight and composition and conjugation efficiency, is the size-exclusion chromatography-multi-angle light scattering detector (SEC-MALS) technique. Herein, we demonstrate the applicability of the SEC-MALS approach for pneumococcal conjugate vaccine product characterization.
Methods:
Capsular polysaccharides for serotypes (STs) 1, 3, 5, 10 A, 18 C, 24 F, and 33 F conjugated to rCRM197 carrier protein were chosen for this study.
Results:
The technique was very straightforward, with a high degree of accuracy (>90% based on standards) and repeatability (<2% RSD) for conjugate molar mass measurements. In addition, leveraging the capability of SEC-MALS for compositional analysis, we were able to get detailed information on the molecular assembly and conformation of the conjugates and further tweak the conjugation process to yield conjugates of a desired molar mass.
Conclusions:
Thus, this study highlights the usefulness of the SEC-MALS technique for in-depth conjugate vaccine biophysical characterization, which is critical for achieving optimal product attributes, driving manufacturing consistency and vaccine potency.
