PolyRMC, Tulane Center for Polymer Reaction Monitoring and Characterization

Recently acquired lab space

Aerial view of Tulane campus

Motto:

Value and impact based on scientific and technical excellence, integrity, and relevance

Mission statement:

To be the world’s premier center for R&D polymerization reaction monitoring

http://tulane.edu/sse/polyRMC PolyRMC is a non-profit entity

Founded in Summer 2007

Wayne F. Reed, Founding DirectorAlina M. Alb, Associate Director for ResearchMichael F. Drenski, Associate Director for Instrumentation Alex Reed, Assistant Director for Operations

Tightly focused but broadly applicable

:

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.

Polymer ‘born characterized’

‘on-command’ polymers

Monitoring and control

• new polymeric materials • medical applications• nanotechnology • new high performance materials

Resins, Paints, Coatings

Industrial R&D and problem solving

Fundamental and applied research

Accelerate R&D of new materials

Advanced characterization

Multi-detector SEC analysis:Multi-angle Light Scattering,

Viscometer, RI, UV detectors

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 products.

R&D, product development, and problem solving in the polymer/pharmaceutical/ natural products industries

• failure to meet product grade specifications; • inconsistency of product quality;• product instabilities, such as precipitation, degradation, or phase separation;• Defects in the products, such as colloids, particulates, and undesirable colors;

Some of PolyRMC’s Initiatives

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.

◦ Deep expertise broadly applicable to applied polymer issues; creativity; advanced instrumentation base; entrepreneurial energy

◦ 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:

- gelatin/oligosaccharide phase separation

- aggregation, degradation, micro-gelation, dry powder dissolution, polymerization - characterization of natural products; xanthan, pectin, gum arabic, alginates;

- multi-detector SEC characterization; - origin and detection of polymer product anomalies; - determination of physical/chemical processes in production of copolymers; - characterization of water soluble polymers for water purification, paints, cosmetics, food;

PolyRMC Expertise

e.g.

• Deep and powerful expertise in highly focused but broadly applicable areas

• Ability to conceptualize problems in general, far-reaching terms

• 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.

Advantages

Types of reactions that we monitor• Synthetic polymerization reactions: free radical, ‘living’,

polycondensation, homogeneous and emulsion phase, in batch, semi-batch, and continuous reactors

• 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, microgelation

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.

Equilibrium characterization of polymer solutions

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 general.

• Multi-detector Size Exclusion Chromatography (SEC), a standard method• Automatic Continuous Mixing (ACM), characterize complex, multicomponent solutions along selected composition gradients. A PolyRMC method.

Methods and techniques used for reaction monitoring and characterization

Achtung!

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.

Do not use equilibrium characterization methods to characterize non-equilibrium systems.

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

viscosifyingemulsifying

bulk

Analyzed gum arabic SEC data

Determining the molecular origins of how a natural product works to emulsify and thicken alimentary

products.

Dextran has a small population of very high mass chains causing separation:

Mn=1,600g/mole

Mw=12,500g/mole

SEC: Origin of oligosaccharide/gelatin phase separation

It’s the oligosaccharide, not the gelatin!

- Seen in SEC light scattering

RI

& L

S90

o (ar

b. u

nits

)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 processes

Light Scattering and Degradation

Signatures for time dependent light scattering enzymatic degradation of linear molecules with different numbers of strands

time [104 sec]

Degradation by laminarinase

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

sidechain strippingrandom chain cleavagerandom backbone cleavage

Proteoglycan ‘monomer’

glycosaminoglycan sidechains

protein backbone

- 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 ScatteringSMSLS: High throughput screening

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.

A typical SMSLS prototype with both flow and batch cells

SMSLS scheme for automatic, continuous monitoring of protein aggregation

NxM protein samples under any desired set of T, pH, concentration, agitation, ionic strength

Mw vs. time for all NxMsamples, continuously and simultaneously on the computer screen

NxM protein samples under any desired set of T, pH, concentration, agitation, ionic strength

Mw vs. time for all NxMsamples, continuously and simultaneously on the computer screen

Note, for aggregating systems that become turbid M =1

M= # of series cells

N= # of parallel cell banks

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

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.

Developing and delivering complete SMSLS systems to Pharmaceutical companies;

How technology transfer will work

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

Therapeutic protein aggregation monitored by SMSLS

Map

p. /

Map

p., t

=0

t (h)

-at ionic strength:1.56 – 50mM

Protein aggregation

All solutions are unstable over time

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 Mass.

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.

0

2 1010

4 1010

6 1010

8 1010

1 1011

0 2000 4000 6000 8000 1 104 1.2 104

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

1/v

1/c

1/v=1/vmax

+Km

/cvmax

Vmax

=1.6x10-10 M/s

Kmax

=0.00142 cm3/g

Michaelis-Menten-Henri Enzyme kinetics

Rapid determination of enzyme kinetics

Hyaluronate degradation by hyaluronidase

Enzymatic degradation monitored by SMSLS

Dissolution of polymers and time release studies

* Small population of aggregates present in dry powder* Aggregates dissolve in time

Dissolution of a polyelectrolyte

Polystyrene sulfonate

Dissolution of dry polysaccharides

Origin of poor dissolution due to formation of aggregates

0

0.05

0.1

0.15

0.2

0.25

0 1 2 3 4 5 6 7 8

Rele

sed

am

ou

nt

dru

g(m

g)

Time (h)

(2)

(1)

(b)

OCH2CHCH2NHCH(CH3)2.HCl

OH (a)

Ethylene Glycol DimethacrylateN-isopropylacrylamide

AcrylonitrileCN

Monitoring drug release by nanohydrogels

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.

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.

Monitoring heterogeneous solutions of polymers and colloids; e.g. proteins amidst bacteria.

Heterogeneous Time Dependent Static Light Scattering (HTDSLS)

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

Applications of Heterogeneous Time Dependent Static Light Scattering (HTDSLS)

HTDSLS: Good data from a classically intractable case of high particulate contamination:

0

500

1000

1500

I(m

V)

0 50 100 150 200 250 300 350

t(s)

[PVP] F(1/s) . 1.5 mg/ml 0.4561BL 856mV...1 mg/ml 0.3205BL 630mV.0.75 mg/ml 0.3596BL 513mV....0.25 mg/ml 0.4328BL 213mV...water 0.4333BL 25mV av 0.40 + 14%

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.21.4x10-6

1.6x10-6

1.8x10-6

2.0x10-6

2.2x10-6

2.4x10-6

2.6x10-6

2.8x10-6

3.0x10-6

3.2x10-6

3.4x10-6

Kc/

I

sin 2̂(/2) + 100*c

Mw=6.1x105 g/mol A2=3.34x10-4mL-mol/g2, Rg=460 A

Schimanowsky, Strelitzki Mullin, Reed, Macromolecules 32, 7055, 1999

5200, 2 micron latex spheres/mL

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)

Fundamental studies of polymerization kinetics and mechanisms

Optimization of reactions at bench and pilot plant levels

Full scale, feedback control of industrial reactors

Automatic Continuous Online Monitoring of Polymerization reactions: 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 interactions.

Principle of ACOMP

ACOMP ‘front-end’: Extraction/dilution/conditioning

ACOMP ‘back-end’: Detector train

UV detector

Refractive indexdetector

Viscometer

Light scattering

Solvent

Reactor

Recent ACOMP advances Copolymerization

Predictive control

Heterogeneous phase; emulsion and inverse emulsion

Living-type polymerization

Continuous reactors

About ACOMP- Monitor important characteristics of

polymerization reactions while they are occurring

- Develop new polymeric materials, understand kinetics and mechanisms.

- Optimize reactions at bench and pilot plant level.

- Full feedback control of large scale reactors:Increased energy efficiencyMore efficient use of non-renewable resources, plant and personnel timeLess emissions and pollution

Stem the flight of manufacturing overseas: Jobs. ACOMP lab. unit

Left: polymer Mw and r vs. conversion; Right: particle size distribution and specific surface area

Raw data and analysis for free radical polymerization of MMA in emulsion at 70C.

- first simultaneous online monitoring of both polymer and particle properties

A. M Alb, W. F Reed, Macromolecules, 41, 2008

Emulsion Polymerization: Example of raw data and analysis

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 reactions

• Monitor processes for completion, unusual events, specific thresholds of product stimuli responsiveness, etc.

• Produce products that consistently meet or exceed specifications.

• These capabilities can be used in the discovery, development, formulation, and quality control stages