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					PolyRMC, Tulane Center for Polymer
Reaction Monitoring and Characterization
                                                        Founded in Summer 2007

                                         Mission statement:
                                    To be the world’s premier center for
                                    R&D     polymerization     reaction
                                    Value and impact based on scientific
                                    and technical excellence, integrity,
 Recently acquired lab space
                                    and relevance
Wayne F. Reed, Founding Director
Alina M. Alb, Associate Director for
Michael F. Drenski, Associate Director
for Instrumentation
Alex Reed, Assistant Director for
                                                    Aerial view of Tulane campus                        PolyRMC is a non-profit entity
                     : Tightly focused but broadly applicable

                                                              SEC analysis:
                                                              Multi-angle Light Scattering,
                                                              Viscometer, RI, UV detectors
                              Industrial R&D        Advanced characterization
                              and problem
                                                        Monitoring and control
                              solving                                            ‘on-command’
   Resins, Paints, Coatings

                                                        Accelerate R&D
                                                        of new materials
                                Fundamental and applied research
                                                        • new polymeric materials
                                                        • medical applications
                                                        • nanotechnology
                                                        • new high performance materials
                         Polymer ‘born characterized’

Through PolyRMC personnel’s many years of industrial collaboration the process of
dealing with confidentiality, intellectual property rights, and all other legal issues has
been streamlined to produce rapid agreements on desirable terms for industries.
Some of PolyRMC’s Initiatives

Fast Turn-around polymer characterization
  • Reduce bottlenecks and lengthy turn around time in workflows.
  • The services can be used to prioritize and complement each industry’s own
  in-house characterization efforts.
  • Set standards of quality control and product reproducibility leading to
  higher efficiency, and establish means of characterizing and improving new

R&D, product development, and problem solving in
the polymer/pharmaceutical/ natural products
  • failure to meet product grade specifications;
  • inconsistency of product quality;
  • product instabilities, such as precipitation, degradation, or phase
  • Defects in the products, such as colloids, particulates, and undesirable
Development of natural products
• Release of proteins, polysaccharides, and other components
during extraction, including enzymatic
activation/deactivation processes.

• Monitoring chemical and physical changes when natural
products are modified by chemical, thermal, radiative, or
enzymatic treatments.

• Determining the types of micro- and nanostructures that can
be formed from natural products.

• Measurement of encapsulation and time-release properties
of natural products used with pharmacological, food, and
other substances.
PolyRMC Expertise
 ◦ Deep expertise broadly applicable to applied polymer issues;
   creativity; advanced instrumentation base; entrepreneurial
 ◦ Adapting our approaches to the many complex processing aspects
   of natural products; extraction, enzymatic modifications,
   chemical tailoring, encapsulation, etc.

 ◦ A track record of success in solving problems:
e.g. - gelatin/oligosaccharide phase separation
   - aggregation, degradation, micro-gelation, dry powder dissolution,
   - characterization of natural products; xanthan, pectin, gum arabic,
   - multi-detector SEC characterization;
   - origin and detection of polymer product anomalies;
   - determination of physical/chemical processes in production of
   - characterization of water soluble polymers for water purification,
 paints, cosmetics, food;

•   Deep and powerful expertise in highly focused but broadly
    applicable areas
•   Ability to conceptualize problems in general, far-reaching
•   Complete, state-of-the-art instrumentation and skills within a
    ‘clean’ university environment
•   Ready access to many online resources
•   Chance to outsource research and problem solving without the
    overhead investment
•   PolyRMC is used to dealing with industrial partners and their
    concerns for IP rights, confidentiality, etc.
   Types of reactions that we monitor
• Synthetic polymerization reactions: free radical, ‘living’, polycondensation,
homogeneous and emulsion phase, in batch, semi-batch, and continuous
• Postpolymerization reactions: hydrolysis, PEGylation, ‘click’ reactions,
grafting, amination, etc.
• Modifications of natural products, especially polysaccharides
• Poplypeptide synthesis reactions
• Oligonucleotide synthesis reactions
• Polymer degradation reactions due to enzymes, chemical agents, heat,
radiation, acids, bases, etc.
• Protein aggregation and other instabilities
• Phase separation, microgelation
• Kinetics of interacting components in complex solutions
• Dissolution of dry powders, emulsions, pastes, etc.
• Release of encapsulated and associated agents
• Production or hydrolysis of polymers amidst bacterial populations
The types of quantities and events that we
monitor during these reactions
   Evolution of polymer molecular weight
   Reaction kinetics; e.g. polymerization, degradation, aggregation rates
   Particle size distributions
   Degree of reaction completion
   Tracing residual monomers and other reagents
   Monomeric and comonomeric conversion
   Reactivity ratios
   Composition drift and distribution
   Intrinsic viscosity
   Unusual or unexpected events during reactions; onset of turbulence,
   Attainment of desired properties, such as stimuli responsiveness;
    ability to encapsulate drugs or other agents, micellization or other
    supramolecular structuration, solubility changes, ability to interact or
    not interact with specific agents, etc.
Methods and techniques used for reaction
monitoring and characterization
   Equilibrium characterization of polymer solutions
• Multi-detector Size Exclusion Chromatography (SEC), a
standard method
• Automatic Continuous Mixing (ACM), characterize complex,
multicomponent solutions along selected composition gradients. A
PolyRMC method.
 Non-equilibrium characterization; PolyRMC methods

• ACOMP (Automatic Continuous Monitoring of
Polymerization reactions); Monitor synthetic reactions, polypeptide
synthesis, polymer modifications, etc.
• Heterogeneous Time Dependent Static Light Scattering
(HTDSLS); Characterize co-existing populations of polymer and
colloids; e.g. bacteria and polymers

• Simultaneous Multiple Sample Light Scattering (SMSLS);
high throughput screening of protein aggregation, solution stability in
                      Do not use equilibrium characterization
Achtung!              methods to characterize non-
                      equilibrium systems.

Many biological polymers in aqueous solutions are inherently
unstable and can aggregate, form microgels, precipitate, or otherwise
degrade in time.
• The time for such instabilities may be seconds, hours, days, even
months or longer.
• It is hence imperative to know if such a solution is in equilibrium, or
at least in a long lived metastable state, before making equilibrium
measurements, such as chromatographic determinations, or single
scattering or other measurements.
• This is why we have developed a number of methods, briefly outlined
below, SMSLS, ACOMP, ACM, and HTDSLS, for monitoring the
kinetics and characteristics of non-equilibrium processes.
• Unfortunately, many researchers spend a lot of time making
measurements on kinetically unstable systems, leading to
irreproducible results and confusion.
Multi-detector size exclusion
chromatography; to be used when
the polymer solution is in equilibrium
 Example of state-of-the art multi-detector Size
 Exclusion Chromatography
Determining the molecular origins of how a natural product works
                    to emulsify and thicken alimentary products.

                     Analyzed gum arabic SEC data

             bulk                                   viscosifying
  SEC: Origin of oligosaccharide/gelatin phase
            It’s the oligosaccharide, not the gelatin!
Dextran has a small population of very high mass chains causing
- Seen in SEC light scattering

                             RI & LS90o (arb. units)

Mn controls the sensation
of sweetness, and
determines commodity
price, but Mw controls
phase separation. This
approach provides a means
of screening this highly
variable natural product.
Monitoring polymer degradation
Light Scattering and Degradation
Signatures for time dependent light scattering
enzymatic degradation of linear molecules with different
numbers of strands

                                     Degradation by laminarinase

           time [104 sec]

 Beta glucan is a
 mixture of double
 and triple strands
  New signatures for time-dependent light scattering
  degradation of branched polymers; determination of
  polymer architecture, kinetics, modes of cleavage
                                                   glycosaminoglycan sidechains
                                                  protein backbone

 Proteoglycan ‘monomer’                           sidechain stripping
                                                  random chain cleavage
                                                  random backbone cleavage

- sidechain stripping, backbone intact

- random sidechain degradation, backbone intact

- sidechain stripping and backbone degradation
Simultaneous Multiple Sample Light
Scattering (SMSLS); when high
throughput and/or long term solution
stability screening is important
Simultaneous Multiple Sample Light Scattering
SMSLS: High throughput screening

                                                      A typical SMSLS
                                                      prototype with both
                                                      flow and batch cells

  A single instrument can monitor stability and reactions of many
  different samples for hours, days, months, automatically, and with
  a single computer; e.g. Monitor protein aggregation.
      SMSLS scheme for automatic, continuous
      monitoring of protein aggregation

                                                               N= # of parallel cell

M= # of series cells

 NxM protein samples under         Mw vs. time for all NxM
 any desired set of T, pH,         samples, continuously and
 concentration, agitation, ionic   simultaneously on the
 strength                          computer screen

 Note, for aggregating systems that become turbid M =1
   Developing and delivering complete SMSLS
   systems to Pharmaceutical companies;
                      How technology transfer will work

PolyRMC will run assays on systems determined by pharmaceutical
sector colleagues, e.g. protein solution stability under a matrix of

If SMSLS proves useful for a given pharmaceutical sector collaborator,
PolyRMC, or associated entity, will build and deliver a turn-key,
customized SMSLS instrument and associated software for the
collaborating company.

PolyRMC also provides an as-needed access service to SMSLS assays,
and related problem solving, in cases where the company might not
need an instrument of its own.
Online monitoring/characterization
of aggregation processes
   Gelatin aggregation

Aggregation process for gelatin solutions at different temperatures,
monitored by ACOMP
   Protein aggregation

   Therapeutic protein aggregation monitored by SMSLS

All solutions are unstable
over time                                         -at ionic strength:
                                                  1.56 – 50mM

                             Mapp. / Mapp., t=0

                                                                        t (h)
     Ranked methods for monitoring and
     quantifying protein aggregation

  Most important aspect of aggregation is change
  in Mass
1. Static Light Scattering:Absolute, model-independent
   change in Mw at the slightest change
       SMSLS: for high throughput

2. Dynamic Light Scattering: Runner-up. Model
   dependent, sensitive to <D>z, only indirectly sensitive to
3. Low angle Mie scattering/diffraction: e.g. Master Sizer. Misses
   the boat. Reports aggregation only after very advanced. Gives
   ‘size’ not mass.
4. Fluorescence. Indirect, insensitive, but better than nothing.
Enzymatic degradation monitored by SMSLS

Hyaluronate degradation by hyaluronidase

                                      Rapid determination of
                                      enzyme kinetics

                                    Michaelis-Menten-Henri Enzyme
                                                          1/v=1/v                +K /cv
                                                                            max        m          max

                                           1 1011

                                                          y = 6.2873e+09 + 8.9157e+06x R= 0.99696
                                           8 10

                                           6 10

                                           4 10

                                                                                   Vmax=1.6x10-10 M/s
                                           2 1010
                                                                                   Kmax=0.00142 cm3/g

                                                      0    2000      4000       6000       8000     1 104   1.2 104
Dissolution of polymers
and time release studies
   Dissolution of a polyelectrolyte
* Small population of aggregates present in dry powder
* Aggregates dissolve in time

                                            Polystyrene sulfonate
Dissolution of dry polysaccharides

Origin of poor dissolution due to formation of aggregates
 Monitoring drug release by nanohydrogels
Poly(acrylonitrile-co-Nisopropylacrylamide), p(AN-c-NIPAM) core-
shell hydrogel nanoparticles were synthesized by microemulsion
polymerization and their feasibility as drug carrier was investigated.
The release of propanolol, PPL from core-shell p(AN-c-NIPAM) 1” and
amidoximated p(AN-c-NIPAM) “2” was continuously monitored by
UV detection with ACM.
                                                                                OH                   (a)



                                   Relesed amount drug(mg)
           Ethylene Glycol                                    0.1                                     (1)

           N-isopropylacrylamide                             0.05

                                                                    0   1   2   3      4     5   6   7      8
                                                                                    Time (h)
Monitoring heterogeneous solutions of
polymers and colloids; e.g. proteins
amidst bacteria.
Heterogeneous Time Dependent Static
Light Scattering (HTDSLS)
Applications of Heterogeneous Time Dependent
Static Light Scattering (HTDSLS)

  HTDSLS: Use flow to create countable scattering peaks from
  colloidal particles, while simultaneously monitoring the
  background scattering due to co-existing polymers

 Determine large particle densities amid polymer chains; e.g.
 spherulites, microgels, bacteria, crystallites, etc.

 Follow evolution of large particles; e.g. in biotechnology reactors
 where bacteria/polymers co-exist. e.g. xanthan productions,
 degradation of polysaccharides, other fermentation reactions

 Permits useful characterization of polymers in solutions which, up
 until now, would be considered far too contaminated with dust and
 other scatterers.
          HTDSLS: Good data from a classically intractable case of
          high particulate contamination:


          5200, 2 micron latex spheres/mL
                                                                                                                                                          [ PV P]                         F
                                                                                             1000                                                         .
                                                                                                                                                          1. 5 m g/ m l                  0.

                                                                                    I(mV )
                                                                                                                                                          BL 856m V    .
                                                                                                                                                          1 m g/ m l                      0
                                                                                                                                                          BL 630m V  .
                                                                                                                                                          0. 75 m g/ m l                0. 3
                                                                                                                                                          BL 513m V  .
                                                                                              500                                                         .
                                                                                                                                                          0. 25 m g/ m l                0. 4
                                                                                                                                                          BL 213m V    .
                                                                                                                                                          w er                             0
                                                                                                                                                          BL 25m V           av 0. 40 + 14%
                                                                                                     0        50    100         150         200        250             300          350
       3.2x10-6                                                                                                                    t(s )



       2.4x10-6                                                                                          Mw=6.1x105 g/mol

                                                                                                         mol/g2, Rg=460 A


                  0.0   0.1   0.2   0.3   0.4   0.5   0.6   0.7   0.8   0.9   1.0       1.1    1.2                        Schimanowsky, Strelitzki Mullin, Reed,
                                           sin^2( /2) + 100*c                                                            Macromolecules 32, 7055, 1999
Heterogeneous time dependent static light
scattering (HTDSLS)
 Co-existing E. Coli and PVP polymers in solution

                               Schimanowsky, Strelitzki Mullin, Reed, Macromolecules 32, 7055, 1999
Automatic Continuous Online
Monitoring of Polymerization
reactions (ACOMP)
Automatic Continuous Online Monitoring
of Polymerization reactions: ACOMP

   Fundamental studies of polymerization
    kinetics and mechanisms

   Optimization of reactions at bench and
    pilot plant levels

   Full scale, feedback control of industrial
Principle of ACOMP

Continuously extract and dilute viscous reactor liquid producing
a stream through the detectors so dilute that detector signals are
dominated by the properties of single polymers, not their
                      ACOMP ‘front-end’:       ACOMP ‘back-end’:
                      Extraction/dilution/co       Detector train
                                                 Light scattering


                                                 Refractive index

                                                   UV detector
                                  - Monitor important characteristics of polymerization
About ACOMP                                          reactions while they are occurring

 - Develop new polymeric materials, understand
     kinetics and mechanisms.
 - Optimize reactions at bench and pilot plant
 - Full feedback control of large scale reactors:
    Increased energy efficiency
    More efficient use of non-renewable
    resources, plant and personnel time
    Less emissions and pollution
 Stem the flight of manufacturing overseas: Jobs.                     ACOMP lab. unit

Recent ACOMP advances
    Copolymerization
    Predictive control
    Heterogeneous phase; emulsion and
     inverse emulsion
    Living-type polymerization
    Continuous reactors
       Emulsion Polymerization: Example of raw data and analysis
    - first simultaneous online monitoring of both polymer and particle
    Raw data and analysis for free radical polymerization of MMA in emulsion at 70C.

Left: polymer Mw and hr vs. conversion; Right: particle size distribution and specific surface area
                                                                                               A. M Alb, W. F Reed, Macromolecules, 41, 2008
Summary: PolyRMC works with many pharmaceutical,
    synthetic, and natural product polymers, with a particular
    emphasis on monitoring processes in solutions of these in
    order to
•   Better understand the processes and mechanisms
    involved in producing such polymers
•   Quantitatively control the factors responsible for the
•   Monitor processes for completion, unusual events,
    specific thresholds of product stimuli
    responsiveness, etc.
•    Produce products that consistently meet or exceed
•   These capabilities can be used in the discovery,
    development, formulation, and quality control stages

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