Transcript web redesign presentation to Dr. Rodgers

Innovative Method For Quality Control of
High Molecular Weight Semi-synthetic
Vaccines
Dietmar Tietz, Ph.D., PMP
DJT Consultants
Laurel, Maryland, USA
[email protected]
2nd World Conference on Magic Bullets (Paul Ehrlich II)
Nuremberg 2008
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.
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 timeconsuming.
 Gel filtration was not a useful analytical method,
since vaccine particles were so large that they
appeared in the void volume.
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.
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 nondenatured 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.
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.)
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.
Vaccine II:
Progressive
stripping of
vaccine particle
surfaces
indicates the
presence of
three major
subpopulations
(Tietz et al.).
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
Comparison of Technologies and Costs
Then and Now
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)
Modern technology makes this innovative 2-D method
affordable and much more practical.
Innovative Method For Quality Control of
High Molecular Weight Semi-synthetic
Vaccines
Dietmar Tietz, Ph.D., PMP
DJT Consultants, Laurel, Maryland, USA  Email: [email protected]
Three more slides available for the
discussion of methods!
2nd World Conference on Magic Bullets (Paul Ehrlich II)
Nuremberg 2008
Additional Information
2-D Serwer-type Gels vs. 2-D O’ Farrell Gels
Serwer-type - used for vaccines
O'Farrell-type - for comparison
 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).
 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.

Schematic of the
horizontal
bidirectional
electrophoresis
apparatus with
buffer-submersed
agarose gels (Philip
Serwer)

Iso-size and iso-freemobility nomogram for the
evaluation of twodimensional gel patterns
(Tietz et al.)

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.
