Presentation on theme: "2nd World Conference on Magic Bullets (Paul Ehrlich II)"— Presentation transcript:
1 2nd World Conference on Magic Bullets (Paul Ehrlich II) Innovative Method For Quality Control of High Molecular Weight Semi-synthetic VaccinesDietmar Tietz, Ph.D., PMPDJT ConsultantsLaurel, Maryland, USA2nd 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 PatternsPatterns 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 parametersResults available within a day or two, not months!Characteristic 2-D patterns for each conjugate preparationUseful for determining vaccine effectiveness and the impact of storage, lyophilization, and sterile filtrationApplicable for any high-molecular weight vaccineand 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 requiredCustom-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 optionNifty, small-footprint electrophoresis equipment with Peltier cooling (~$2k - $5k)Modern technology makes this innovative 2-D method affordable and much more practical.
11 Three more slides available for the discussion of methods! Innovative Method For Quality Control of High Molecular Weight Semi-synthetic VaccinesDietmar Tietz, Ph.D., PMPDJT Consultants, Laurel, Maryland, USA Three more slides available for the discussion of methods!2nd World Conference on Magic Bullets (Paul Ehrlich II)Nuremberg 2008
13 2-D Serwer-type Gels vs. 2-D O’ Farrell Gels Serwer-type - used for vaccinesLower (1st) and higher (2nd) concentration submersed agarose gels used to achieve predominant separation according to charge or size in one direction.Non-denaturing conditionsGels not touchedUsed 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 comparisonIsoelectric focusing and SDS polyacrylamide gel electrophoresis (sieving) used to achieve 2-D separation according to charge and size.Denaturing conditionsTransfer of gel slabUsed for macromolecules with a typical size range of 20 kD kD.
14 Schematic of thehorizontal bidirectional electrophoresis apparatus with buffer-submersed agarose gels (Philip Serwer)Migrate remaining content to the CMS and update as neededContent not extramuralUpdate display of organization structure and staff contact informationStrategic planning reports, testimony,Enhance home page designImplement dynamic sorting of initiatives and conferences to science areasImplement dynamic calendar of events/press releasesOffer access to improved Web site traffic statistics; e.g., program officers can monitor access of their program area pages
15 Iso-size and iso-free-mobility nomogram for the evaluation of two-dimensional gel patterns (Tietz et al.)