Innovative Method For Quality Control of High Molecular Weight Semi-synthetic Vaccines

1. Innovative Method For Quality Control of High Molecular Weight Semi-synthetic Vaccines Dietmar Tietz, Ph.D., PMP DJT Consultants Laurel, Maryland, USA djt@his.com 2nd World Conference on Magic Bullets (Paul Ehrlich II) Nuremberg 2008

2. The Vaccine: High molecular weight protein-polysaccharide conjugated vaccines prepared to protect infants from infections with bacterial meningitis (Haemophilus influenzae type b, Hib). Samples were kindly donated by Robbins and Schneerson (NICHD, NIH, Bethesda). • Bacterial coat polysaccharide particles were obtained by sonication of bacteria. To increase immunogenicity, harvested capsular polysaccharide particles were conjugated with proteins, e.g., toxoided tetanus toxin and Haemophilus protein P2. • Covalent attachment of proteins converts the polysaccharide to a T-cell dependent antigen that triggers a protective immune response in small children.

3. The Challenge: • The effectiveness of earlier vaccine samples was unpredictable. This was likely a result of randomizing steps - sonication & crosslinking - in the preparation of these vaccines. • Immunological testing for activity was very time-consuming. • Gel filtration was not a useful analytical method, since vaccine particles were so large that they appeared in the void volume.

4. EnvisionedMagic BulletSolution: • Fast analytical procedure for predicting vaccine effectiveness based on physical parameters of vaccine particles. • Strategy: • Develop a computer-assisted gel electrophoretic procedure that exploits differences in sample charge and size.

5. 1-D Gel Electrophoresis • We used a horizontal electrophoresis apparatus with buffer-submersed agarose gels (developed by P. Serwer UTHSC, San Antonio, Texas). This apparatus was especially designed for the analysis of very high molecular weight particles such as intact viruses. • The image shows gel patterns of non-denatured meningitis vaccines at different agarose concentrations. The samples yielded an uninterpretable smear, although electrophoretic conditions were appropriate based on co-electrophoresis of samples with narrowly defined particle size distributions.

6. 2-D Gel Patterns • (Serwer apparatus with submersed gels) • (I-III) Non-denatured vaccine preparations, (S) two carboxylated polystyrene samples used for standardization. Standardized by overlay of curvilinear size/charge nomogram (Tietz et al.)

7. 2-D Gel Patterns Patterns of original images that have been transformed from a curvilinear to a rectangular coordinate system of particle size and mobility, which is related to charge (Tietz et al.). • Vaccine I was ineffective. • Vaccines II and III are effective immunogens. • Vaccine II is a mixture of vaccine batches. • Vaccine III is crosslinked with well-defined protein P2.

8. Vaccine II:Progressive stripping of vaccine particle surfaces indicates the presence of three major subpopulations (Tietz et al.).

9. Magic BulletSolution and Biomedical Significance Vaccine quality control based on physical parameters • Results available within a day or two, not months! • Characteristic 2-D patterns for each conjugate preparation • Useful for determining vaccine effectiveness and the impact of storage, lyophilization, and sterile filtration • Applicable for any high-molecular weight vaccine and particles as large as or larger than intact viruses

10. 1983 - 1995: Precision Perkin Elmer microdensitometer ($300k) interfaced with two mainframe computers ($300k) Trained computer operators required Custom-made electrophoresis equipment with bulky cooling systems, pumps, and power supplies (~$30K) Today: Digital camera interfaced with desktop or laptop computer ($2k) Do-it-yourself option Nifty, small-footprint electrophoresis equipment with Peltier cooling (~$2k - $5k) Comparison of Technologies and Costs Then and Now Modern technology makes this innovative 2-D method affordable and much more practical.

11. Innovative Method For Quality Control of High Molecular Weight Semi-synthetic Vaccines Dietmar Tietz, Ph.D., PMP DJT Consultants, Laurel, Maryland, USA  Email: djt@his.com Three more slides available for the discussion of methods! 2nd World Conference on Magic Bullets (Paul Ehrlich II) Nuremberg 2008

12. Additional Information

13. Serwer-type - used for vaccines Lower (1st) and higher (2nd) concentration submersed agarose gels used to achieve predominant separation according to charge or size in one direction. Non-denaturing conditions Gels not touched Used for subcellular-sized particles with a size of 2,000 kD - 2,000,000+ kD (size of intact viruses and larger). O'Farrell-type - for comparison Isoelectric focusing and SDS polyacrylamide gel electrophoresis (sieving) used to achieve 2-D separation according to charge and size. Denaturing conditions Transfer of gel slab Used for macromolecules with a typical size range of 20 kD - 500 kD. 2-D Serwer-type Gels vs. 2-D O’ Farrell Gels 

14. Schematic of the horizontal bidirectional electrophoresis apparatus with buffer-submersed agarose gels (Philip Serwer) 

15. Iso-size and iso-free-mobility nomogram for the evaluation of two-dimensional gel patterns (Tietz et al.) 

16. References • Robbins, JB, Schneerson, R, Anderson, P, Smith, DH, JAMA 1996, 276, 1181–1185. • Tietz, D, Aldroubi, A, Schneerson, R, Unser, M, Chrambach, A, Electrophoresis 1991, 12, 46-54. • Tietz, D, Electrophoresis 2007, 28, 512–524. • Serwer, P, Anal. Biochem. 1985, 144, 172-178. 