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Innovative Method For Quality Control of High Molecular Weight Semi-synthetic Vaccines Dietmar Tietz, Ph.D., PMP DJT Consultants Laurel, Maryland, USA.

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Presentation on theme: "Innovative Method For Quality Control of High Molecular Weight Semi-synthetic Vaccines Dietmar Tietz, Ph.D., PMP DJT Consultants Laurel, Maryland, USA."— Presentation transcript:

1 Innovative Method For Quality Control of High Molecular Weight Semi-synthetic Vaccines Dietmar Tietz, Ph.D., PMP DJT Consultants Laurel, Maryland, USA 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 Envisioned Magic Bullet Solution:  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 Bullet Solution 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 Comparison of Technologies and Costs Then and Now :  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) 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  Three more slides available for the discussion of methods! 2nd World Conference on Magic Bullets (Paul Ehrlich II) Nuremberg 2008

12 Additional Information

13 2-D Serwer-type Gels vs. 2-D O’ Farrell Gels 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 kD. 

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,  Tietz, D, Electrophoresis 2007, 28, 512–524.  Serwer, P, Anal. Biochem. 1985, 144, 


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