Poly RMC Tulane Center for Polymer Reaction Monitoring

Poly. RMC, 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 monitoring Motto: Recently acquired lab space Value and impact based on scientific and technical excellence, integrity, and relevance Wayne F. Reed, Founding Director Alina M. Alb, Associate Director for Research Michael F. Drenski, Associate Director for Instrumentation Alex Reed, Assistant Director for Operations http: //tulane. edu/sse/poly. RMC Aerial view of Tulane campus Poly. RMC is a non-profit entity

: Tightly focused but broadly applicable Multi-detector SEC analysis: Resins, Paints, Coatings Industrial R&D and problem solving Multi-angle Light Scattering, Viscometer, RI, UV detectors Advanced characterization Monitoring and control ‘on-command’ polymers Accelerate R&D of new materials Fundamental and applied research • new polymeric materials • medical applications • nanotechnology • new high performance materials Polymer ‘born characterized’ Through Poly. RMC 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 Poly. RMC’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 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;

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.

Poly. RMC Expertise ◦ 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: e. g. - 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;

Advantages • 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 • Poly. RMC 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 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.

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 Poly. RMC method. Non-equilibrium characterization; Poly. RMC 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.

Achtung! Do not use equilibrium characterization methods to characterize nonequilibrium 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 emulsifying

SEC: Origin of oligosaccharide/gelatin phase separation It’s the oligosaccharide, not the gelatin! Dextran has a small population of very high mass chains causing separation: Mn=1, 600 g/mole Mw=12, 500 g/mole 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. RI & LS 90 o (arb. units) - Seen in SEC light scattering

Monitoring polymer degradation processes

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, backbone intact - random sidechain degradation, backbone intact - sidechain stripping and backbone degradation sidechain stripping random chain cleavage random backbone cleavage

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 banks M= # of series cells Note, for aggregating systems that become turbid M =1

Developing and delivering complete SMSLS systems to Pharmaceutical companies; How technology transfer will work Poly. RMC 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, Poly. RMC, or associated entity, will build and deliver a turn-key, customized SMSLS instrument and associated software for the collaborating company. Poly. RMC 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 -at ionic strength: 1. 56 – 50 m. M Mapp. / Mapp. , t=0 All solutions are unstable over time 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 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.

Enzymatic degradation monitored by SMSLS Hyaluronate degradation by hyaluronidase Rapid determination of enzyme kinetics Michaelis-Menten-Henri Enzyme kinetics

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) coreshell 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. (a) (b) CN Acrylonitrile Ethylene Glycol Dimethacrylate N-isopropylacrylamide

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/m. L Mw=6. 1 x 105 g/mol A 2=3. 34 x 10 -4 m. Lmol/g 2, Rg=460 A Schimanowsky, Strelitzki Mullin, Reed, 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 reactors

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 interactions. ACOMP ‘front-end’: Extraction/dilution/co nditioning ACOMP ‘back-end’: Detector train Light scattering Reactor Viscometer Refractive index detector Solvent UV detector

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 efficiency More efficient use of non-renewable resources, plant and personnel time Less emissions and pollution Stem the flight of manufacturing overseas: Jobs. Recent ACOMP advances Copolymerization Predictive control Heterogeneous phase; emulsion and inverse emulsion Living-type polymerization Continuous reactors ACOMP lab. unit

Emulsion Polymerization: Example of raw data and analysis - first simultaneous online monitoring of both polymer and particle properties Raw data and analysis for free radical polymerization of MMA in emulsion at 70 C. 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: Poly. RMC 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