Devices, systems, and methods for non-contact stirring of samples

Abstract

Described herein devices and systems for performing non-contact mixing of solutions during spectroscopic measurement, as well as methods of using thereof.

Description

Attorney Docket No.11656-005WO1 DEVICES, SYSTEMS, AND METHODS FOR NON-CONTACT STIRRING OF SAMPLES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority of U.S. Provisional Application No.63 / 639,399, filed April 26, 2024, which is hereby incorporated herein by reference in its entirety. BACKGROUND

[0002] It is often necessary in chemistry and related fields to mechanically mix solutions containing two or more components. Examples include, but are not limited to, solutions containing polymers, colloids, salts, chelating agents, surfactants, excipients, denaturing agents, catalysts, initiators, and solutions in which agents are being dissolved or suspended. Such mixing can be used to produce a homogenous multicomponent solution much more quickly than a solution that has no agitation and relies on diffusion, which is an extremely slow process. A simple example of this is the dissolution of a sugar cube in a cup of tea or coffee. If the cube is allowed to simply sit in the solution, without convection, it can take hours to dissolve, whereas, with agitation it can be made to dissolve and uniformly disperse sugar molecules within seconds.

[0003] Modes of mechanical agitation include, but are not limited to, stirring, oscillating convection, shaking, bubbling, and reciprocating mechanical action. As far as stirring, it is commonplace to stir solutions using a magnetic stir bar placed in the bottom of a vessel containing a solution. The solution can then be stirred by applying an external magnetic field to the stir solution. However, this type of contact-stir can degrade complex molecules present in the solution, such as proteins (e.g., monoclonal antibodies), polysaccharides, vaccines, and colloids (e.g., lipid nanoparticles, liposomes, and viral capsids). Further, many biologic drugs and therapeutic agents are subject to degradation by contact-stirring. The degradation often takes the form of aggregation of the molecules or colloids, or, conversely, the breaking apart of aggregates, colloids, or complexes. This degradation cam negatively impact the ability of researchers to study such materials in solution.

[0004] Improved methods of mixing solutions containing such components offers the ability to improve the analysis of complex systems. The devices, systems, and methods described herein address these and other needs.Attorney Docket No.11656-005WO1 SUMMARY

[0005] It is general practice in chemistry and related fields to mechanically agitate liquids in which one or more agents are dissolved or suspended, in order to much more quickly achieve a homogeneous solution or suspension, than by relying upon diffusion. Such agitation can take several forms, including stirring, oscillatory stirring, shaking, reciprocal motion, and blending. Described herein are devices, systems, and methods for the non- contact stirring of samples. In contrast to devices, systems, and methods in which contact stirring is employed, when stirring occurs without contact (e.g. with a stirrer suspended in the solution), components of the solution (e.g., biomolecules and / or colloids) experience little or no degradation.

[0006] The devices and systems described herein can produce convection within a fluid present in a receptacle using a motor attached integrally to a cap assembly for a receptacle, which allows non-contact stirring of solutions. In some embodiments, the devices described herein can be sized to be received within a spectroscopic cuvette. In this way, the devices can provide for non-contact stirring within spectroscopic cuvettes and other receptacles. Optionally, the device can further comprise one or more access ports (e.g., one access port, two access ports, or three access ports) that can be utilized to add and / or remove components from the receptacle while the device remains seated in the receptacle and stirring a solution in the receptacle.

[0007] The inclusion of access ports allows users to conduct a wide variety of titration and dilution processes. These processes can be carried out and monitored continuously and automatically (e.g., using syringe pumps to add or remove components from the receptacle). The inlet and outlet ports can be used to perform measurements involving discrete or continuous addition and discrete or continuous removal of liquid from the receptacle. These latter processes are particularly useful when the composition of the solution is changing due to addition of components by discrete addition, continuous pumping, dialysis, and other membrane-mediated processes. As a non-contact stirrer, the devices described herein can be particularly useful when agitating molecules, macromolecules, and colloids that can degrade when contact stir is used. These substances include biologic drugs and therapeutic agents.

[0008] Accordingly, in some aspects, provided herein are devices that comprise a motor coupled integrally to a drive shaft and impeller, wherein the drive shaft and the impeller are insertable within a receptacle such that the impeller can deliver non-contactAttorney Docket No.11656-005WO1 stirring or reciprocal motion to a fluid present within the receptacle when driven by the motor.

[0009] In some aspects, the motor comprises a stepper motor electric motor, a non- stepper electric motor, an oscillating motor, or a reciprocating motor.

[0010] In some aspects, the device further comprises a cap assembly comprising a body portion insertable within an opening of the receptacle. In certain aspects, the motor is mounted on the cap assembly. In certain aspects, the drive shaft extends from the motor through the body portion and into the receptacle when the body portion is inserted within the opening of the receptacle. In some embodiments, the receptacle comprises a cuvette, and the length of the drive shaft extending from a bottom of the body portion of the cap assembly is selected such that the impeller and drive shaft to not interfere with spectroscopic measurements performed within the cuvette.

[0011] In some aspects, the drive shaft is detachably coupled to the motor.

[0012] In some aspects, the device can further comprise a mounting post (118) extending from a bottom of the body portion, wherein the mounting post is configured for attachment of a membrane. In certain aspects, the mounting post can be configured for hermetically sealed attachment of a membrane. In some aspects, the cap assembly allows spectroscopic monitoring of membrane mediated processes. In certain aspects, the membrane mediated process comprises membrane dialysis.

[0013] In some aspects the device can be positionable within a spectroscopic instrument that performs spectroscopic measurements, either continuously or at intervals. In certain aspects, the spectroscopic instrument can be chosen from static and dynamic light scattering, UV / visible absorption, infra-red absorption, fluorescence, Raman scattering, circular dichroism, circular birefringence, polarimeter, dielectric spectrometer, and microwave absorption.

[0014] In some aspects, the fluid in the receptacle (e.g., being non-contact stirred) can comprise a biologic drug in molecular, macromolecular, or colloidal form.

[0015] In some aspects, the device is used in conjunction with an electrode assembly (120). In some aspects, the electrode assembly can reside independently of the device in the receptacle. In other aspects, the electrode assembly is integral to the device and resides inside the receptacle. In other aspects, the electrode assembly is external to the receptacle.

[0016] In some aspects, the cap assembly further comprises one or more access ports (116) that allow solids, liquids, gases, or a combination thereof to be added to and / or removed from the receptacle. In some aspects, the cap assembly comprises a single accessAttorney Docket No.11656-005WO1 port. In other aspects, the cap assembly comprises two access ports. In other aspects, the cap assembly comprises three aspects. In certain aspects, the one or more access ports have a cross-sectional area of from 1% to 20% of a cross-sectional area of an opening of the receptacle.

[0017] In some aspects, the receptacle comprises a spectroscopic cuvette. In certain aspects, the cuvette has an optical path length of 1 cm or less. In certain aspects, the receptacle is optically transparent within the UV and / or visible region. In certain aspects, the receptacle comprises a quartz cuvette, a borosilicate cuvette, or a plastic cuvette.

[0018] In some aspects, systems described herein can comprise a device described herein and a receptacle, such as a spectroscopic cuvette. In some aspects, the systems can further comprise one or more of: a controller for operating the motor, a spectrometer, one or more syringe pumps for adding and / or removing fluids from the receptacle via one or more access ports present in the device, or a combination thereof.

[0019] Also provided herein are methods of using the devices and systems described herein. In some aspects, methods described herein can comprise adding a sample comprising an analyte to a receptacle; inserting the device described herein within the receptacle such that the impeller can deliver non-contact stirring or reciprocal motion to the sample present within the receptacle when driven by the motor; and stirring the sample with the device. In certain aspects, the method further comprises performing one or more spectroscopic measurements of the sample.

[0020] In some aspects, the device further comprises one or more access ports that allow solids, liquids, gases, or a combination thereof to be added to and / or removed from the receptacle; and the method further comprises adding a substance to the receptacle or removing a substance from the receptacle via the one or more access ports.

[0021] In some aspects, the substance is added or removed via the one or more access ports at a continuous rate. In other aspects, the substance is added or removed via the one or more access ports in one or more aliquots.

[0022] In some aspects, the method further comprises performing one or more spectroscopic measurements of the sample prior to addition of the substance or the removal of the substance from the receptacle. Such measurements can serve as a baseline against which future measurements are judged to assess the impact of the addition of the substance or the removal of the substance from the receptacle.

[0023] In some aspects, the method further comprises performing one or more spectroscopic measurements (e.g., an individual measurement, continuous measurement overAttorney Docket No.11656-005WO1 a period of time), of the sample during and / or after addition of the substance or the removal of the substance from the receptacle.

[0024] In some aspects, the method comprises a spectrophotometric titration.

[0025] In some aspects, the method comprises adding a substance to the receptacle via the one or more access ports, stirring the sample with the device, and performing one or more spectroscopic measurements of the sample to observe a response to the addition of the substance.

[0026] In some aspects, a solvent is flowed through a first access port into the receptacle containing the sample having an analyte therein at a first concentration; and fluid is removed from the receptacle via a second access port at the same rate, thereby continuously reducing the first concentration of the analyte. In some aspects, the method can further comprise spectroscopically monitoring the sample to assess one or more concentration-dependent properties of the analyte. In certain aspects, the one or more concentration-dependent properties of the analyte can comprise weight average molar mass, virial coefficients, electrical conductivity, UV / visible absorption, fluorescence, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figures 1A-1E illustrate an example device described herein. Figure 1A shows a top perspective view of the example device. Figure 1B shows a bottom perspective view of the example device. Figure 1C shows a front view of the example device. Figure 1D shows a top (left) and bottom (right) view of the example device. Figure 1E shows a photograph of an example device operatively positioned within a receptacle (in this example, a spectroscopic cuvette).

[0028] Figures 2A-2E illustrate an example device described herein that includes three 1.5mm ports. Figure 2A shows a top perspective view of the example device. Figure 2B shows a bottom perspective view of the example device. Figure 2C shows a front view of the example device. Figure 2D shows a top (left) and bottom (right) view of the example device. Figure 2E shows a photograph of an example device operatively positioned within a receptacle (in this example, a spectroscopic cuvette).

[0029] Figures 3A-3E illustrate an example device described herein that includes a mounting post extending from a bottom of the body portion and configured for attachment of a membrane. Figure 3A shows a top perspective view of the example device. Figure 3B shows a bottom perspective view of the example device. Figure 3C shows a front view of theAttorney Docket No.11656-005WO1 example device. Figure 3D shows a top (left) and bottom (right) view of the example device. Figure 3E shows a photograph of an example device operatively positioned within a receptacle (in this example, a spectroscopic cuvette).

[0030] Figures 4A-4D illustrate an example device described herein that includes an integrated electrode assembly. Figure 4A shows a top perspective view of the example device. Figure 4B shows a bottom perspective view of the example device. Figure 4C shows a front view of the example device. Figure 4D shows a top (left) and bottom (right) view of the example device.

[0031] Figure 5 shows a photograph of an example device including both an electrode assembly and a mounting post configured for attachment of a membrane operatively positioned within a receptacle (in this example, a spectroscopic cuvette).

[0032] Figures 6A-6C illustrate an example device described herein that includes an integrated 45 degree offset electrode assembly. Figure 6A shows a top perspective view of the example device. Figure 6B shows a bottom perspective view of the example device. Figure 6C shows a front view of the example device.

[0033] Figure 7 is a plot illustrating the deleterious impact of contact stirring on a sample. As shown in Figure 7, a monoclonal antibody that was contact stirred quickly aggregated, whereas the non-contact stir sample (labeled ‘Suspended Stir’) shows no aggregation over 24 hours. Mw / M0is the relative molar mass determined by total intensity light scattering during the stir periods. In both cases the stirring was at 500 RPM.

[0034] Figure 8 is a photograph comparing samples of samples stirred by contact stirring and non-contact stirring. On the left monoclonal antibody solutions are seen to be still transparent after 24 hours of non-contact stir at 500RPM. On the right, monoclonal antibody solutions are seen to be cloudy, due to contact-stir induced aggregation, after 24 hours of contact-stir at 500 RPM.

[0035] Figure 9 shows an overlay of Size Exclusion Chromatography chromatograms for non-contact stirred and contact-stirred samples of a monoclonal antibody after 24 hours at 500RPM. Despite the strong aggregation in the contact-stirred sample, no difference was observed between the SEC chromatograms. This indicates that (i) the aggregates comprise only a very small mass fraction of the antibody population, (ii) the aggregates are either blocked or broken up by the SEC column, and / or (iii) SEC does not detect this type of strong aggregation.

[0036] Figure 10A shows Debye plots for determination of Mw and A2 (i) using continuous addition and extraction of solvent to polymer solution in cuvette, at constantAttorney Docket No.11656-005WO1 volume, and (ii) using discrete aliquots of solvent added through the access port to the cuvette, wherein the volume changes in this case. Continuous non-contact stir from the device ensured rapid and efficient mixing as solvent was added in both (i) and (ii). R was the measured Rayleigh Ratio, Cpthe polymer concentration, and K the usual optical constant for vertically polarized incident light.

[0037] Figure 10B shows that data from the continuous and discrete Debye plots of an oligopeptide show non-ideal behavior, seen in the non-linear form of the data. The representation is in R / K, where R is the measured Rayleigh Ratio of the scattered light, and K is an optical constant, independent of oligopeptide concentration.

[0038] Figure 11 shows the relative scattering from a solution of sodium dodecyl sulfate, originally at 11.8mM in aqueous 10mM NaCl and diluted continuously with pure aqueous 10mM NaCl. Two runs are shown and yield a CMC of 7.88mM + / - 1.9%.

[0039] Figure 12 is a plot showing the hydrodynamic diameter, DH, of 28nm diameter latex spheres in water.400 measurements made with the following repeated cycle: 45s stir, 15s for fluid in cuvette to come to rest, 60s DLS measurement with a Brookhaven Instruments BI-90. A Phidgets 3315_0 unipolar stepper motor was programmed to achieve the stir / no-stir cycle.

[0040] Figure 13 is a plot showing the conductivity (arbitrary units) versus [NaCl] using the cuvette electrode assembly. The non-ideality of the conductivity is seen by the curvature. The measurement covers 1M NaCl to 0, and uses a syringe pump to flow pure water into the access port, and a second syringe pump to withdraw fluid from the cuvette.at the same rate.

[0041] Figure 14 shows the absorbance of fluorescein in 50mM NaCl aqueous solution at 600nm as it is automatically diluted from 40 mg / ml to 1 mg / ml with pure 50mM NaCl aqueous solution via a syringe pump at 0.002 ml / s, while liquid is withdrawn from the cuvette at the same rate, thus keeping the cuvette volume constant at 2 ml.

[0042] Figure 15 schematically illustrates the circumstances at play when utilizing a device described herein with three access ports.

[0043] Figure 16 shows the normalized intensity of light scattered from a 1ml sample of 0.2mg / ml Na-Hyaluronate (HA) solution originally in a cuvette at [NaCl]=0. A 100mM NaCl aqueous solution is infused into the cuvette with a syringe, together with a 0.4mg / ml HA solution in [NaCl]=0 in a separate syringe, both syringes actuated by a Chemyx Nexus 6000 dual-head syringe pump at a rate, each, of 0.002 ml / s, while a separate Nexus pump withdraws cuvette solution at 0.004 ml / s, thus keeping the concentration of HA constant inAttorney Docket No.11656-005WO1 the cuvette at 0.2 mg / ml, while maintaining the cuvette volume at 1ml. There is a 3.5x increase in light scattering due to the NaCl shielding the negative charges on the HA polyion, thus reducing HA interchain repulsion and increasing osmotic compressibility, and hence also increasing light scattering intensity.

[0044] Figure 17 is a plot of the fraction of original concentration of 0.010 g / ml of polymer in the cuvette vs time, with a fixed volume of 1ml and flow rate of 0.001 ml / s, from a reservoir of 20ml.

[0045] Figure 18A shows the light scattering intensity during dissolution of dried gelatin crystals when introduced into water at 40oC, non-contact stirred at 500RPM, at a concentration of 1 mg / ml. The dissolution is complete by 15 minutes, seen by the leveling off of the light scattering intensity.

[0046] Figure 18B shows the weight average molar mass, Mw, versus time while bovine serum albumen (BSA) is non-contact stirred at 500 RPM at T=25oC, at a concentration of 5 mg / ml, into a 5mM PBS buffer at pH=7.4. Detailed Description Definitions

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0048] Fluid: A liquid, which may be pure, or have dissolved or suspended components. For example, a fluid may be a pure solvent such as, but not limiting, water, dimethylsulfoxide (DMSO), toluene, tetrahydrofuran, acetone, methanol, ethanol, carbon disulfide, and many other pure solvents. The pure fluid may be a mixture of two or more solvents. The pure fluid may have dissolved components such as, but not limiting, ions, small neutral molecules, surfactants, dyes, fluorescence markers, chelation agents, buffers, oligomers, and polymers of biological or synthetic origin. Suspended materials in the fluid include, for example, whole cells, clusters of cells, organelles from cells, micelles, liposomes,Attorney Docket No.11656-005WO1 vesicles, nanoemulsions, microemulsions, emulsions, surfactant stabilized structures, quantum dots, metal sols, and combinations thereof.

[0049] Access port: An aperture in the integral device that allows adding liquids, solids, or gas to the solution in the receptacle (e.g., cuvette) and also allows withdrawal of liquid in the receptacle. For liquids and gases, the access port can be made to accommodate the outer diameter of a syringe needle, or for tubing, such as stainless steel, Teflon, Nylon, Peek, etc.. A septum or covering may also be included in the access port to seal the contents from the environment. In embodiments that use dialysis membranes with the device there are inlet and outlet flow paths for the circulating dialysate. In these, the inlet and outlet access ports provide access for adding and removing material, such that no additional access ports are needed.

[0050] Biologic drug: Any medical or therapeutic agent derived from biological sources, or synthesized by humans, such as engineered proteins, DNA, RNA, and polysaccharides. Biologic drugs, also termed ‘biologic medicine products’, include, but are not limited to, monoclonal antibodies, polyclonal antibodies, any type of protein in general, polysaccharides, polynucleic acids, including all forms of RNA and DNA, viral capsids, agents used to encapsulate drugs or therapeutic agents, such as viral capsids, liposomes, and synthetic polymers and copolymers, vaccines, and agents composed of weakened or deactivated viruses, and combinations of proteins, polysaccharides, lipids, nucleic acids, and other components.

[0051] Chromatographic method: Includes any of several well-known methods, such as, but not limited, to, gel permeation chromatography, size exclusion chromatography, high pressure liquid chromatography, field flow fractionation chromatography, interaction chromatography, multi-dimensional gel permeation chromatography, and gas chromatography.

[0052] Colloid: Any material that can be suspended in a liquid. Examples include micelles, liposomes, vesicles, emulsions, microemulsions, semiconductor particles, nanoparticles of any type, oxide particles, cell organelles and fragments. The term ‘polymer’ will also imply ‘colloid’ throughout, as well as any type of biologic agent.

[0053] Polymer: A molecule consisting of at least ten monomeric units, whether of the same monomer, or different monomers. The term ‘polymer’ subsumes ‘colloid’ throughout, as well as all biologic products, oligonucleotides, oligosaccharides, and oligopeptides.Attorney Docket No.11656-005WO1

[0054] Convection: Movement of a liquid through space, as opposed to the liquid being stationary in space or undergoing diffusion or osmosis.

[0055] Contact-stirring: the use of a magnetic stir bar or other stirring device which makes contact with the cuvette or vessel inner surface while providing the stirring motion.

[0056] Non-contact stirring: Stirring a solution in a cuvette or vessel without the stirring device making contact with the cuvette inner surface while providing the stirring, oscillating, reciprocating, or other form of motion.

[0057] Receptacle: A fluid containment vessel. In some embodiments, receptacle can be dimensioned so as to be reversibly insertable with a spectroscopic instrument. In some embodiments, the receptacle can be a square cuvette made of glass, quartz, or a transparent polymer. In other embodiments, its shape can be circular, polygonal, elliptical, or irregular.

[0058] Cuvette: A device for containing a solution that is reversibly insertable within a spectroscopic instrument. This can be of any shape, such as, but not limited to, cylindrical, square, polygonal, and a tapered body. It can be made of any material that provides optical transparency at the points of radiation entry and detection of the cuvette. The cuvette can be made of any transparent material, such as glass, quartz, polymer, and plastic. It can also be made of any non-transparent material, as long as it has a transparent aperture(s) providing optical transparency at the points of radiation entry and detection of the cuvette. Such materials can include, but are not limited to, opaque polymer or plastic, metal, ceramic, and composite materials. Where a non-spectroscopic method is used, e.g. conductivity, pH probes, the cuvette need not be optically transparent, nor have any transparent apertures.

[0059] Optical transparency. A material which allows passage of most or all of the incident electromagnetic radiation incident upon it, over a desired wavelength range.

[0060] Electromagnetic Radiation: Includes electromagnetic radiation in any part of the spectrum, from gamma rays, through to x-rays, ultraviolet, visible, infrared, microwave, and radio waves.

[0061] Spectroscopic instrument: Any instrument capable of making a measurement on a sample, which involves providing incident electromagnetic radiation on the sample and detecting electromagnetic radiation emanating from the sample as a result of the incident radiation. Examples include, but are not limited to, static and dynamic light scattering, Raman scattering, Ultraviolet and visible absorption, fluorescence, circular dichroism, circular birefringence, polarimetry, infra-red absorption, microwave absorption.

[0062] A spectroscopic instrument can also include a cell containing electrodes that can provide electric fields of varying frequency. Such cells are used, for example, inAttorney Docket No.11656-005WO1 dielectric spectroscopy where the electrical response of a solution, for example its impedance is measured over a range of frequencies.

[0063] Convection means: This includes any means for driving convection in a cuvette. It includes, but is not limited to, rotational stirring, oscillatory stirring, reciprocal plunging, bubbling gas, and shaking.

[0064] Motor: This includes any type of electrical motor, such as an externally controlled stepper motor, simple d.c. and a.c. motors, and oscillating and reciprocating motors. In the case of motors requiring external control, a control means is connected reversibly to the motor. Where the motor requires only a voltage or current source, this can be supplied by a battery integral to the device assembly, or provided by an external d.c. or a.c. source.

[0065] Drive shaft: Refers to a shaft of any shape or material that can be coupled to the motor and either has an integral impeller, or can be coupled to an impeller.

[0066] Impeller: This can be of any shape that produces convection in the liquid in the cuvette. It can be shaped to provide any desired fluid flow field. It may have one or more blades with no angle, or angled in such as way as to provide vertical convection. In some embodiments, the impeller can be formed from a non-conductive material (e.g., Teflon). In some embodiments, the entirety of the drive shaft and impeller in contact with a fluid present in the receptacle are formed from a non-conductive material (e.g., Teflon) and / or are coated with an electrically insulating coating.

[0067] Static light scattering: The measuring of the scattered intensity from a solution or suspension in a cuvette. The measured intensity can be treated either as a relative intensity, such as, but not limited to, the intensity scattered from the sample initially, or at some other point during a process, or as an absolute intensity, which allows molar mass determination of the scatterers in the solution or suspension.

[0068] Thermodynamic method: Includes, but not limited to, thermogravimetric analysis, Differential scanning calorimetry, and isothermal calorimetry.

[0069] Dynamic light scattering: The measuring of fluctuations in scattered intensity or electric field, autocorrelated to yield an autocorrelation function. The autocorrelation function can be analyzed according to standard methods to determine the scatterers’ translational or rotational diffusion coefficient, or distributions of these diffusion coefficients. The methods currently employed also provide a measure of dispersity of the scatterers. The relative or absolute scattered intensity can also be simultaneously measured, as in the case of static light scattering.Attorney Docket No.11656-005WO1

[0070] Cap Assembly: A cap which can fit onto the receptacle in which the first fluid and second fluid are separated by a membrane. The cap assembly may be a simple cap that fits onto the receptacle. Alternatively, the cap assembly may include additional components to facilitate containment and / or manipulation of the first fluid and / or the second fluid, positioning of the membrane, or measurement / interrogation of the first fluid and / or the second fluid. For example, the cap assembly may be a cap with apertures to allow conductors attached to the electrodes, such as wires, to pass from the electrodes (whether integral with the cap assembly or present within a separate electrode assembly) and through the cap for external electrical connections. The cap assembly may include an integral electrode assembly that fits into the receptacle. The cap assembly may include a means of stirring the contents of the receptacle, whether the electrode assembly is integral with the rest of the cap assembly, or resides inside the receptacle independently of the cap assembly, or resides external to the receptacle. The cap assembly may contain a means of affixing a membrane that divides the solution within the receptacle into a first fluid and a second fluid. This latter aspect may contain and combine any of the previously mentioned features. The cap assembly may contain one or more access ports that allow introduction of any solution with any components and can also include one or more access ports for withdrawing liquid from the receptacle.

[0071] Electrode: Any of a pair of conductors which can be placed in a fluid in the receptacle and which can receive voltage or current from a.c. or d.c. voltage or current sources. a.c. voltage is preferably used, as it reduces or eliminates electrode polarization, electrolytic reactions, build-up or plating on the electrodes. The current is transmitted between the electrodes by ions or other charged particles between the electrodes. The voltage or current source may be gated on and off intermittently, via a programmable switch or relay. The elecrodes may also be shorted out intermittently via a programmable switch or relay. The conductors can be made from any conducting material, such as, but not limited to metals, graphite, graphene, glassy carbon, carbon nanotubes, certain ceramics (e.g. zirconia), conductive polymers (e.g. polyanaline and polypyrrole), doped silicon, and metal oxides (e.g. indium tin oxide, titanium dioxide, etc.). The electrodes can take forms such as sheets or plates, wire meshes, wires, cylinders, or other specialized forms. The electrodes can be connected to the voltage or current source by wires or other conductors (e.g. metallic strips) affixed to the electrodes which lead out of the receptacle. The electrodes can reside in the receptacle for i) measuring conductivity of fluid in the receptacle and ii) applying electric fields strong enough to affect polymers. If the electrodes are disposed outside the receptacle they can be used for applying electric fields strong enough to affect polymers.Attorney Docket No.11656-005WO1

[0072] Electrode assembly: Refers to the means in which the electrodes are disposed within the receptacle. The electrodes may be mounted on a cage-like or box-like device which inserts into the receptacle. The electrodes may also be mounted on a device containing a means of providing convection for the fluid, such as stirring. The electrodes may also be mounted on a device that allows separation of Fluid 1 from Fluid 2 by a membrane. The electrodes may also be mounted on a device that allows for both convection of the fluid, such as stirring, and separation of Fluid 1 from Fluid 2 by a membrane. The electrodes may also be free-standing within the receptacle, not attached to any other device in the receptacle. The electrodes may also fit into custom recesses in the walls of the receptacle. The electrodes may also be manufactured integrally with the receptacles. In the case where the electrodes are free standing, are affixed to the receptacle walls, or are integrated into the receptacle in the manufacturing process the term ‘electrode assembly’ is intended to include this. This latter case may also be termed ‘means of disposition of the electrodes within the receptacle’.

[0073] Electrode support: A structure on which one or more electrodes are disposed or attached. The electrode support can be fabricated from any suitable material. In some cases, the electrode support is fabricated from a conductive material, such as a metal. If desired, the electrode support is fabricated from the same material(s) as the one or more electrodes disposed or attached to the electrode support. In other embodiments, the electrode support can be fabricated from a polymer.

[0074] Membrane: Any type of membrane, whether open or closed, through which matter can pass, including, but not limited to, fluids, small molecules, ions, gases, polymers, colloids, and specific types of fluids, such as various polar and non-polar fluids. Examples, not limiting, include membranes made of polyvinylidene difluoride (PVDF), polyimide, polyamides (e.g., nylons and aramids), carboxymethylcellulose, hybrid membranes such as hyaluronic acid / carboxymethylcellulose, polyethersulphone, cellulose acetate, cellulose nitrate, sintered metals, gels, polycarbonate, polysulfone polytetrafluoroethylene (PTFE, sometimes called by one of its commercial names ‘Teflon’). Examples of membranes used in medical dialysis include Theranova 400, Theralite 2100, Revaclear 400, Polyflux 17L. The Membrane may also comprise a biological tissue, such as, but not limited to, skin, muscle, fat, neurons, and bone. Membranes used for ion batteries can include, among others, polymer based membranes (e.g. polyethylene oxide, Nafion, polyacrylonitrile, as well as ceramic and composite membranes, graphene oxide, gel electrolyte membranes, and ionic liquid-based membranes.Attorney Docket No.11656-005WO1

[0075] Membrane mediated process. This includes any process wherein a system is divided into two parts, separated by a membrane, and the transfer of chemical species, mass, or electrical charge occur. This includes membranes used in dialysis, electrodialysis, fuel cells, batteries and other electrical storage devices, size filters. The mediation may occur due to diffusion, osmosis, reverse osmosis, electrical potentials and fields, active transport assisted by molecules such as ATP. Devices and Systems

[0076] The devices and systems described herein can produce convection within a fluid present in a receptacle using a motor attached integrally to a cap assembly for a receptacle, which allows non-contact stirring of solutions. In some embodiments, the devices described herein can be sized to be received within a spectroscopic cuvette. In this way, the devices can provide for non-contact stirring within spectroscopic cuvettes and other receptacles. Optionally, the device can further comprise one or more access ports (e.g., one access port, two access ports, or three access ports) that can be utilized to add and / or remove components from the receptacle while the device remains seated in the receptacle and stirring a solution in the receptacle.

[0077] The devices described herein can be used to spectroscopically monitor multi- component solutions while convection is provided by non-contact stirring, oscillating or reciprocal motion, in a cuvette, with a particular emphasis on how molecules and colloids behave as solution conditions change. These solution conditions include, but are not limited to, changes in ionic strength, pH, concentration of one or more species, types of solutes, such as different types and valences of salts, surfactants, chelating agents, and other excipients, change of temperature or pressure, and dissolution of dry agents, such as lyophilized macromolecules or colloids.

[0078] In some rare cases spectroscopic instruments have sample holders with magnetic drive. An example of this is the simultaneous multiple sample light scattering (SMSLS) instrument ARGEN, from Yokogawa Fluence Analytics (Houston, Texas). In such cases it is possible to arrange a shaft with stirring impeller in the cap of the cuvette and drive it with the instrument’s built-in magnetic drive, thus providing non-contact stirring. When such magnetic drive capabilities are absent, as in the majority of commercial spectroscopic instruments, it is possible to position an external motor above the cuvette, and couple it to the drive shaft and impeller, and thus drive the impeller and shaft in this way. However, this approach requires precise alignment of the external motor with the shaft assembly, andAttorney Docket No.11656-005WO1 requires external support structures to fix the motor above the cuvette. Additionally, it requires time and labor to perform the alignment and support operations.

[0079] There are currently several commercially available options for stirring cuvettes magnetically, using stirrer cages, and external magnetic drives. However, these all involve contact-stirring, which is to be explicitly avoided in light of the detrimental effects it has on macromolecules and colloids, and, in particular, on biologic drugs and therapeutic agents.

[0080] The devices described herein provide the drive unit, shaft, and impeller in an integral unit which seat directly within a cuvette (e.g., within the top opening of the cuvette), without need for alignment and support between the drive motor and the shaft / impeller assembly. The devices can provide non-contact stirring or reciprocal motion to the solution in the cuvette.

[0081] By way of example, referring now to Figures 1A-1E, 2A-2E, 3A-3E, 4A-4D, 5, and 6A-6C, in some embodiments, the device (100) can comprise a motor (102) coupled integrally to a drive shaft (104) and impeller (106). The drive shaft (104) and the impeller (106) are insertable within a receptacle (108) such that the impeller (106) can deliver non- contact stirring or reciprocal motion to a fluid (110) present within the receptacle when driven by the motor (102).

[0082] In some embodiments, the device further comprises a cap assembly (112) comprising a body portion (114) insertable within an opening of the receptacle.

[0083] The motor (102) can comprise any suitable motor that can drive rotation of the impeller. By way of example, in some embodiments, the motor comprises a stepper motor electric motor, a non-stepper electric motor, an oscillating motor, or a reciprocating motor. In certain embodiments, the motor comprises a stepper motor.

[0084] In some embodiments, the motor (102) is mounted on the cap assembly (112). For example, in some embodiments, the motor can be mounted one or above a top surface of the cap assembly.

[0085] The drive shaft (104) can extend from the motor (102) through the body portion (114) of the cap assembly (112) and into the receptacle when the body portion is inserted within the opening of the receptacle.

[0086] In some embodiments, the drive shaft is detachably coupled to the motor. In some embodiments, the impeller is detachably coupled to the drive shaft. In these embodiments, the drive shaft and / or impeller can be readily removed and replaced should theAttorney Docket No.11656-005WO1 drive shaft and / or impeller need to be cleaned, or if the drive shaft and / or impeller are corroded or otherwise damaged from contact with solutions.

[0087] In some embodiments, the the receptacle comprises a spectroscopic cuvette. In certain embodiments, the cuvette has an optical path length of 1 cm or less. In certain embodiments, the receptacle comprises a quartz cuvette, a borosilicate cuvette, or a plastic cuvette. In certain embodiments, the receptacle is optically transparent within the UV and / or visible region.

[0088] In some embodiments, the device is positionable within a spectroscopic instrument that performs spectroscopic measurements, either continuously or at intervals. The spectroscopic instrument can be chosen from, for example, static and dynamic light scattering, UV / visible absorption, infra-red absorption, fluorescence, Raman scattering, circular dichroism, circular birefringence, polarimeter, dielectric spectrometer, and microwave absorption.

[0089] In some embodiments, the fluid / sample present in the receptacle (and stirred by the device) comprises a biologic drug in molecular, macromolecular, or colloidal form.

[0090] Referring now to Figures 3A-3E, in some embodiments, the device further comprises a mounting post (118) extending from a bottom of the body portion (114). The mounting post can be configured for attachment of a membrane. In certain embodiments, the mounting post can be configured for hermetically sealed attachment of a membrane

[0091] In some embodiments, the cap assembly allows spectroscopic monitoring of membrane mediated processes (e.g., membrane dialysis).

[0092] Referring now to Figures 4A-4D and 6A-6C, in some embodiments, the device is used in conjunction with an electrode assembly (120). In some embodiments, the electrode assembly resides independently of the device in the receptacle. In other embodiments, the electrode assembly is integral to the device and resides inside the receptacle (as shown in Figures 4A-4D and 6A-6C). In other embodiments, the electrode assembly is external to the receptacle.

[0093] Referring now to Figures 2A-2E, in some embodiments, the device further comprises one or more access ports (116) e.g., one access port, two access ports, or three access ports) that allow solids, liquids, gases, or a combination thereof to be added to and / or removed from the receptacle. The access ports can be of varying dimensions. In some embodiments, the one or more access ports (in combination) have a cross-sectional area of from 1% to 20% of a cross-sectional area of an opening of the receptacle.Attorney Docket No.11656-005WO1

[0094] Also described herein are systems that comprise a device described herein and a receptacle, such as a spectroscopic cuvette. In some embodiment, the systems can further comprise one or more of: a controller for operating the motor, a spectrometer, one or more syringe pumps for adding and / or removing fluids from the receptacle via one or more access ports present in the device, or a combination thereof. Methods

[0095] Also described herein are methods of using the stir a fluid present within a receptacle. These methods can comprise adding a sample comprising an analyte to a receptacle; inserting a device described herein within the receptacle such that the impeller can deliver non-contact stirring or reciprocal motion to the sample present within the receptacle when driven by the motor; and stirring the sample with the device.

[0096] In some embodiments, the method further comprises performing one or more spectroscopic measurements of the sample.

[0097] In some embodiments, the device further comprises one or more access ports that allow solids, liquids, gases, or a combination thereof to be added to and / or removed from the receptacle; and the method further comprises adding a substance to the receptacle or removing a substance from the receptacle via the one or more access ports.

[0098] As discussed above, the one or more access ports can comprise apertures formed within the device that allow for substances (liquids, solids, and / or gases) to be introduced into and / or withdrawn from the receptacle without removing the device from the receptacle. In this way, substances can be introduced and / or withdrawn while stirring is maintained and / or while spectroscopic measurements are performed.

[0099] Depending upon the nature of studies performed, a wide range of samples may be introduced into and / or withdrawn from the receptacle. Examples of solids that may be added to the receptacle include, but are not limited to, powders or crystals such as salts, sugars, or any other agent in dry form. Examples of liquids that may be added to the receptacle include, but are not limited to, solvents for diluting the contents of the receptacle, solutions for varying ionic strength (e.g., by adding an electrolyte solution), solutions for varying the pH (e.g., by adding an acidic or basic solution), a solution comprising a reagent, reactant, or other component which interacts with a component present in the receptacle, and a liquid (solvent or non-solvent) different from liquids already present in the receptacle. Examples of gases that may be added to the receptacle include, but are not limited to, inert gases, such as Argon or Nitrogen, added to facilitate the progress of certain reactions, such asAttorney Docket No.11656-005WO1 free radical polymerization, and active gases, such as oxygen, added to quell a chemical reaction or promote a chemical reaction.

[0100] Examples of liquids that can be removed from the receptacle include, but are not limited to, liquid aliquots removed for analysis by some other means, such as another spectroscopic method, a chromatographic method, a thermodynamic method, pyrolysis, atomic absorption, viscometry, or other types of analysis. The withdrawal of aliquots can also be used to subsequently add more liquid to the cuvette to dilute the contents. In some cases where liquids or gases are added or remove, the access port can be dimensioned to accommodate a syringe needle. In such cases, a septum or other means of sealing the needle access port from entry or escape of gas when the needle is not present in the access port. If solids are to be introduced into the receptacle, then the access port can be larger than needle size to facilitate addition of a solid.

[0101] In some embodiments, the one or more access ports can be used to introduce aliquots of a liquid to the original contents of the receptacle, where the original contents contain a solvent and an analyte of interest, such as a polymer or colloid. For example, if pure solvent is added the concentration of the polymer or colloid will decrease upon adding each aliquot. If desired, in some embodiments, once the cuvette volume is reached (e.g., an initial volume of 1 mL has been increased to a limit of 3mL), then an access port can be used to remove a desired amount of liquid (e.g., 2mL by way of example), so that further dilutions can be made. This process can be repeated as many times as desired to allow for continued dilution (and spectroscopic characterization of) the sample.

[0102] Such methods can be used to perform a variety of analyses, including by way of example: the use of static light scattering to determine the weight average molar mass Mw, and also the second virial coefficient, A2, and potentially higher virial coefficients, such as the third virial coefficient A3, or higher order virial coefficients of, for example, a polymer or other macromolecule; the use of dilution in combination with dynamic light scattering to determine the polymer or colloid diffusion coefficient D as a function of concentration, to obtain both the self-diffusion coefficient D0and the hydrodynamic interaction parameter kD, in the expression D=D0(1+kDc+ ….), where c is the polymer or colloid concentration; to determine the critical micelle concentration, CMC, of any micelle forming molecule, such as sodium dodecyl sulfate (SDS), or self-micellizing copolymers, such asAttorney Docket No.11656-005WO1 poly(ethylene glycol)-b-poly(lactic acid) and poly(ethylene glycol)-b-poly(D,L-lactide-co- glycolide); to determine the association and dissociation behavior of any system where mass- action allows such associations and dissociations, such as in viral capsid assembly, polymer coacervation, colloidal nanoparticle self-assembly, and Pickering emulsions; to use an electrode assembly in the receptacle to measure conductivity versus concentration of any type of electrolyte (virtually all of these show non-ideal, non-linear behavior as concentration increases significantly); association and dissociation under this type of dilution may also be made while monitoring with UV / visible spectroscopy, or fluorimetry; and assessing the kinetics of dissolution.

[0103] In some embodiments, the device can include a second access port, a third access port, or more access ports. In some of these embodiments, a first access port can be used for introducing liquid or solid materials to the receptacle, while a second access port can be used to remove liquid from the receptacle.

[0104] Some examples of methods in which multiple access ports may be used include, but are not limited to, continuous flow systems: by holding the receptacle volume constant, while adding an external liquid continuously to dilute an analyte (e.g., a polymer or colloid) present in a solution initially present in the receptacle, and removing liquid at an equal rate, a continuous record of the analyte concentration behavior can be obtained. This can enable continuous automatic records instead of the discrete additions and discrete data points. The receptacle volume can be held constant by matching the pump furnishing the inlet fluid to the pump extracting fluid from the receptacle. Another option is to provide a withdrawal tube connected to the withdrawal pump, which can be fixed at a desired height within the receptacle, thus automatically ensuring constant receptacle volume; titration of the initial receptacle sample containing analyte (e.g., a polymer or colloid) with any agent, including, but not limited to, salts, including organic salts, denaturants, surfactants, acids, bases, chelating agents, oligopeptides, oligonucleotides, other polymers or colloids, enzymes, nanoparticles of any type, excipients, and ionic liquids; if it is desired to hold the analyte (e.g., a polymer or colloid) concentration constant, or have it change more slowly during situations, a concentrated analyte (e.g., a polymer or colloid) solution can be added through one inlet port, the titrating agent through another inlet port, and the withdrawal port is a third access port.Attorney Docket No.11656-005WO1

[0105] In some cases, it may be useful to have three or more ports, such as the situation just mentioned, allowing for two or more liquids to be added to the receptacle during an experiment. For example, not limiting, a solvent, such as a buffer, different than the solvent originally in the receptacle can be added, while another substance, such as an electrolyte, denaturant, acid or base, is added through the other port.

[0106] These situations can also be run in the other sense; i.e., one can start with no analyte (e.g., a polymer or colloid), and add the analyte continuously. This can be particularly useful to test for the reversibility of systems that have concentration dependent associations.

[0107] The pumps used for the continuous flow systems can be of any type normally used in millifluidic scenarios (e.g. HPLC, GPC, SEC, etc). These include syringe pumps, peristaltic pumps, piston pumps, high pressure liquid chromatography pumps, gear pumps, and slot pumps.

[0108] Detectors external to the spectroscopic measuring instrument can also be placed in the outlet or inlet flows. These detectors can include any type of detector equipped with a flow cell, such as DLS, SLS, UV / visible absorption, IR absorption, fluorescence, Refractivity, viscosity, and conductivity.

[0109] In some embodiments, the device further comprises one or more access ports that allow solids, liquids, gases, or a combination thereof to be added to and / or removed from the receptacle; and the method further comprises adding a substance to the receptacle or removing a substance from the receptacle via the one or more access ports.

[0110] In some embodiment, the substance is added or removed via the one or more access ports at a continuous rate. In other embodiments, the substance is added or removed via the one or more access ports in one or more aliquots.

[0111] In some embodiments, the method further comprises performing one or more spectroscopic measurements of the sample prior to addition of the substance or the removal of the substance from the receptacle. Such measurements can serve as a baseline against which future measurements are judged to assess the impact of the addition of the substance or the removal of the substance from the receptacle.

[0112] In some embodiments, the method further comprises performing one or more spectroscopic measurements of the sample during and / or after addition of the substance or the removal of the substance from the receptacle.

[0113] In some embodiments, the method comprises a spectrophotometric titration. In some embodiments, the method comprises adding a substance to the receptacle via the one or more access ports, stirring the sample with the device, and performing one or moreAttorney Docket No.11656-005WO1 spectroscopic measurements of the sample to observe a response to the addition of the substance.

[0114] In some embodiments, a solvent is flowed through a first access port into the receptacle containing the sample having an analyte therein at a first concentration; and fluid is removed from the receptacle via a second access port at the same rate, thereby continuously reducing the first concentration of the analyte.

[0115] In some embodiments, methods can further comprise spectroscopically monitoring the sample to assess one or more concentration-dependent properties of the analyte. The one or more concentration-dependent properties of the analyte can comprise weight average molar mass, virial coefficients, electrical conductivity, UV / visible absorption, fluorescence, or a combination thereof.

[0116] Optionally, in some embodiments, the devices described herein can be used to facilitate the spectroscopic monitoring of membrane-mediated processes. In such cases, a cuvette can be partitioned with a membrane, such that there is Fluid 1 in the spectroscopically interrogated portion of the cuvette and a Fluid 2, of different composition, is in the other portion of the cuvette. Often it is desirable to have convection in Fluid 1 to provide mixing of the solutes or particles passing from Fluid 2 into Fluid 1. This makes the process much more efficient and repeatable than relying on diffusion alone to mix the components coming into Fluid 1 across the membrane from Fluid 2. The non-contact stirring device can be made integrally into a single unit with the membrane partitioning feature.

[0117] Optionally, in some embodiments, the devices described herein can be used to facilitate the spectroscopic monitoring of solutions in the presence of an applied electric field. Electrodes can be incorporated into the assembly containing the stir device, to produce an integral unit insertable into a receptacle. The non-contact stir device can also be used in conjunction with independent electrode assemblies, the latter of which can be either inside or outside the receptacle. When inside the receptacle, the electrodes can be used (i) at low current to measure conductivity, and hence electrolyte concentration in a fluid, and (ii) to apply medium to large electric fields to analytes (e.g., polymers or colloids) in fluid, to monitor the response of the analytes to the electric fields. When outside the receptacle, the electrodes can be used to apply medium to large electric fields to analytes, but cannot be used for conductivity measurements.

[0118] Alternatively, for measuring concentration of specific ions, solid state sensors, are available in millimeter sizes that can fit into a receptacle. These include pH sensors (H+selective), K+, Ca2+, Cl-, I-, and others. Examples of such sensors include Hach IntellicalAttorney Docket No.11656-005WO1 ISECL 181 Chloride sensor, Hanna Instruments HI4011 Iodide detector, and MI-710 pH probe from Microelectronics, Inc. In some embodiments, these can be inserted within the receptacle via one or more access ports present in the devices described herein. Alternatively, such sensors can be integrated within the devices described herein.

[0119] In some embodiments, the devices, systems, and methods described herein can also be used to guide the formulation of biologic active agents (e.g., biologic drugs) to find concentration regimes of specific formulation components over which the biologic drug formulation is stable. For example, not limiting, the stability of a biologic drug, such as a monoclonal antibody, over a range of concentrations of formulation components can be determined, including pH ranges. These formulation components can include, but are not limited to, electrolytes of different valences and symmetries -e.g. NaCl (monovalent, symmetric), MgCl2 (asymmetric with divalent cation), MgSO4 (symmetric divalent)- surfactants, and any other additives, excipients, and stabilizers.

[0120] Examples of biologic active agents include, but are not limited to, Adalimumab (sold under the trade name Humira), Rituximab (sold under the trade name Rituxan), Etanercept (sold under the trade name Enbrel), Trastuzumab (sold under the trade name Herceptin), Bevacizumab (sold under the trade name Avastin), Infliximab (sold under the trade name Remicade), Insulin glargine injection (sold under the trade name Lantus), Pegfilgrastim (sold under the trade name Neulasta), Interferon beta-1a (sold under the trade name Avonex), Ranibizumab (sold under the trade name Lucentis), insulin, glucagon, glucagon-like peptides such as liraglutides (Victoza, Saxenda) and semaglutides (Ozempic, Wegovy), tirzepatide (Manjaro), interferons, interleukins, hormones, blood factors, recombinant proteins (e.g. erythropoietin), fusion proteins (alefacept), classical viral vaccines, polysaccharide conjugate vaccines, and mRNA / lipid nanoparticle vaccines.

[0121] Aspects of the devices, systems, and methods described herein are related to the devices, systems, and methods described in PCT / US2024 / 026575, filed April 26, 2024, and PCT / US2024 / 052628, filed October 23, 2024, each of which is hereby incorporated by reference in its entirety.

[0122] The examples below are intended to further illustrate certain aspects of the compositions and methods described herein and are not intended to limit the scope of the claims. EXAMPLESAttorney Docket No.11656-005WO1

[0123] The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art.

[0124] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures, and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0125] The following standard test methods were utilized to characterize materials and compositions described herein. Example 1. Comparison of Contact versus Non-Contact Stirring

[0126] Biologic drug damage by contact stirring versus no damage by non- contact stir. To illustrate the importance of the non-contact stir device and methods, we have found that contact-stir damages virtually all biologic drugs, including monoclonal and polyclonal antibodies, lipid nanoparticles, mRNA, DNA, vaccines, polysaccharides, viral capsids, and other biologics.

[0127] By way of example, not limiting, Figure 7 shows the relative molar mass of an antibody under contact stir and non-contact stir (labelled ‘Suspended Stir’ in the figure), as determined by continuous total intensity light scattering. The contact-stirred sample showed immediate and large-scale aggregation, whereas the sample that was non-contact stirred did not aggregate over 24 hours of non-contact stirring.

[0128] Figure 8 shows that the difference between contact-stir and non-contact stir was visible to the eye in this case. The contact-stir samples were visibly cloudy, whereas the non-contact stir samples remain transparent. It is important to note that aggregation in biologic samples is not always visible to the unaided eye. Significant aggregation can occur while the sample remains transparent to the eye.Attorney Docket No.11656-005WO1

[0129] Importantly, Size Exclusion Chromatography (SEC), which is a standard but flawed method for aggregate detection, shows no evidence of the aggregation caused by contact-stir. Figure 9 shows the chromatograms (using UV detection) for the non-contact stirred and contact-stirred solutions after 24 hours at 500 RPM. The chromatograms are staggered in elution volume artificially, in order to show that they are identical. The area under the curves in each case are equal, showing that the large aggregates formed must be a small mass fraction of the contact-stirred sample, and that the aggregates themselves do not show up in the chromatogram; the aggregates were either blocked by the SEC column, or, less probably, broken up by it. Example 2. Use of the Devices and Systems for Non-Contact Stirring and Analysis of Samples.

[0130] Continuous measurement of ideal and non-ideal solution behavior. In ideal systems, such as gases, liquids, solutions of components, and so on, there is no interaction between components, and the behavior of the material is non-linear. For example, the light scattering intensity of an ideal solution, its osmotic pressure, viscosity, etc., all increase linearly with the concentration of the solute when the system is ideal. Otherwise, all these properties become non-linear when there are interactions among particles and the solution is no longer ideal.

[0131] Continuous Debye plot for determination of polymer weight average molar mass, Mw, the second virial coefficient A2, as well as higher virial coefficients A3, A4, etc. The intensity of scattered light, in terms of the Rayleigh Ratio, R, is measured as the concentration of polymer Cp starts at a given value and then decreases continuously by feeding pure solvent into the cuvette through an access port, while withdrawing fluid from the cuvette at the same rate through a second access port, thus keeping the volume in the cuvette constant while diluting the initial polymer solution. The integral stir device ensures that the solvent is rapidly and efficiently mixed during the addition and withdrawal of fluid. In the particular case here, a syringe pump was used to pump pure solvent into the cuvette, and a second syringe pump, with matched flow rate, was used. This set-up is not limiting, as other types of pumps can be used, as well as other constant volume methods, such as putting a fluid withdrawal tube, via a second access port, at any desired fixed height, and hence fixed volume, in the cuvette.

[0132] The Rayleigh Ratio was measured and used to determine Mwand A2of an oligopeptide in aqueous solution with pH=8.15, originally at 0.010 g / cm3, via the well know scattering equationAttorney Docket No.11656-005WO1 ^^^^ =1 ^+ 2^ ^ ^^ + 3^^^ + ⋯.^where Cpis the polymer fixed volume in the cuvetteand the flow rate. K is the optical constant for vertically polarized light ^2^^^^^ / ^^^^^ ^=^^^^

[0133] Figure 10A showsplot result for the oligopeptide, and the resulting Mwand A2. The volume in the cuvette was held constant at 1mL using the matched flow rate of two syringe pumps. Also shown is the result when discrete aliquots of solvent were added through the access port, and withdrawn once the level in the cuvette reached 3mL, at which point fluid was withdrawn through the access port, leaving 1mL of fluid, which was then subject to further aliquot dilution. In both cases the integral stir device ensured quick and efficient mixing. The results for Mw and A2 are in good agreement between the two methods. A Fluence Analytics Argen static light scattering unit was used for the scattering measurements.

[0134] This is a good example of non-ideal behavior. Figure 10B shows the intensity of scattered light versus concentration of oligopeptide is non-linear, with a negative second derivative, due to non-ideal effects, captured by the positive second virial coefficient A2. The solid lines show second order polynomial fits to the data.

[0135] Determination of micellar CMC (critical micelle concentration) at elevated temperature, 45oC, demonstrating use of the device above room temperature, using continuous addition and withdrawal of 10mM NaCl aqueous solution, keeping the cuvette volume constant at 2ml. The flow rate of pure aqueous 10mM NaCl into the cuvette was 0.002ml / s, at a fixed volume of 2ml. The concentration of SDS was initially at 11.57mM. A syringe pump was used to flow the pure aqueous 10mM NaCl into the cuvette through an access port, and a second syringe pump with its rate matched to the first continuously withdrew cuvette fluid through a second access port.

[0136] Figure 11 shows relative scattering intensity, Mw / M0, of sodium dodecyl sulfate (SDS), starting at 1.0 and diminishing as dilution proceeded. The CMC is found where there is a break in the continuous curve. Two separate runs are shown, with a determination of CMC of 7.88mM + / - 1.9%. It is important to note that the data remain clean down to 0.15mM, a nearly 100x dilution from the original concentration. This quality is due to the quick and efficient mixing provided by the integral non-contact stir device.Attorney Docket No.11656-005WO1

[0137] This is another good example where light scattering is non-linear due to the non-ideality of the surfactant system. The plot of Mw / M0, is directly proportional to Rayleigh Scattering Ratio intensity I.

[0138] Use with Dynamic Light Scattering (DLS). When a fluid has no motion the intensity autocorrelation g2(t) function in DLS is governed by the diffusion of the scattering particles ^^ ^ "#$^ ^ = 1 + ewhere Γ= &'^where D is the particles’ mutual diffusion coefficient (which becomes the particle self- diffusion coefficient D0 when G is extrapolated to zero concentration), and q is the magnitude of the scattering vector '⃗ 4^^' =^ sin - / 2where n is the index of refraction of the pure solvent, l is the vacuum wavelength of the incident light, and q is the angle of detection in the scattering plane. When the fluid is in motion with velocity .⃗ there is a Doppler shift in the scattered light, which disturbs the diffusive term, leading to ^^^^ "#$ / 01⃗ ∙31⃗^ = 1 + e eD cannot be extracted from thisdistribution, are precisely known. In flow situations where '⃗ and .⃗ are perpendicular to each other, this term can be eliminated '⃗ ∙ .⃗ = '. cos 9 = 0 ;<= 9 = ^ / 2where f is the angle between '⃗ and .⃗.

[0139] Where the convective motion of the fluid in the stirred cuvette cannot bearranged to make '⃗ ∙ .⃗ = 0, the stir can be periodically stopped so that a DLS measurementcan be made over an interval where the fluid is at rest, typically between 1s-60s. This will allow D to be determined from ^^^^^.

[0140] The stirring can be periodically stopped by stopping the motor with a relay or other type of switch, or by a computer program for programmable pumps. The fluid inlet and removal can continue during the DLS measurement period. If it is desired to not lose portions of the dilution process, the inlet and withdrawal port pumps can be stopped during the DLS measurement, in addition to stoppage of the stir. The pumps can be stopped either by relays or other switches, or by computer program if the pumps are programmable.Attorney Docket No.11656-005WO1

[0141] DLS data under periodic stop-stir.28nm latex spheres in water were used with the stop-stir DLS method, using the non-contact cuvette stir device, with 1ml fluid, in a Brookhaven Instruments BI-90 DLS instrument. The Python code controlling the Phidgets 3316-0 unipolar stepper motor was set to stir the sample for 45 seconds, and 400 measurements were made on the following schedule: The motor stirred at 400 RPM for 45 seconds, then stirring was stopped for 15 seconds to allow motion of the fluid in the cuvette to stop, and then a DLS measurement lasting 60 seconds was made. Figure 12 shows 400 measurements made over 13 hours, yielding a hydrodynamic diameter, DH, of 27.8nm + / -0.44 nm (+ / - 1.6%).

[0142] Electrolyte conductivity versus concentration. The electrical conductivity of electrolytes and polyelectrolytes in solution is usually non-linear as concentration increases. Figure 13 uses the cuvette electrode assembly, in conjunction with the integral non-contact stir device, to measure the conductivity of an aqueous NaCl solution, as [NaCl] decreases as pure water flows in through the access port, and a pump with matched flow rate withdraws the fluid.

[0143] UV absorbance vs concentration for a chromophore. If concentration of a chromophore is high enough it could introduce nonlinearity into the absorption and fluorescence emission spectra.

[0144] Figure 14 shows Visible absorbance of Na-fluorescein at 600nm, starting at 40 mg / ml. At this concentration all UV wavelengths are saturated in a 1cm quartz cuvette, hence the visible wavelength was used. The data show perfect linearity of absorbance versus [fluorescein], demonstrating that the solution is ideal up to 40 mg / ml. The non-contact convection device was used with a feed of 50mM NaCl aqueous solution.

[0145] 3-port and higher multi-port mixing. While the foregoing examples monitor the effects of dilution on a system containing polymers or colloids, there are instances where it is desirable to hold the concentration of the polymer in the receptacle constant, or to vary it along a controlled trajectory as other components are added or subtracted from the fluid. Figure 15 schematically illustrates the circumstances at play when utilizing a device described herein with three access ports. In these cases: Q1= flow rate of substance X from Reservoir 1; electrolyte, denaturant, acid, base, excipient, polymer, colloid, etc. Q2= flow rate of polymer solution from Reservoir 2, of same type of polymer as in the receptacle initiallyAttorney Docket No.11656-005WO1 Q3=flow rate from receptacle to waste or, optionally, through a flow-equipped detector (UV / vis, fluorimeter, conductivity, refractive index, injection into a chromatography column, etc.) CR1,X= concentration of X in Reservoir 1 CR2,p= concentration of polymer (colloid or biologic) in Reservoir 2 V0= initial volume of receptacle V(t)= time-dependent volume in receptacle CX(t)= concentration of X in the receptacle Cp,0= initial concentration of polymer in the receptacle Cp(t)= time-dependent concentration of polymer in the receptacle mp(t)=mass of polymer in the receptacle Flow balance for constant volume in receptacle; Q1+Q2=Q3 The mass in the receptacle changes as >?^ = ^? ?^^J^A^,^C^ − ^E C^, GℎI=I ^^^@^ =where V(t)=V0

[0146] As an example, not limiting, consider the case where polymer concentration in the receptacle Cp(t) is to be held constant, Cp,0, and the receptacle volume is to remain constant at V0. Then, mp(t)=mp,0=Cp,0V0 and constant CKLMp(t)=Cp,0 requiresKJ = 0 = ^A^,^C^ −LMNOC^so that ^ = LMPQ=PQA^,^ NO PRCp,0 PROperationally, establish a desired Q (e.g. 7.2 ml / hou PTUPR1 r), then ^A^,^ = C^,F PRAnd establish Q2(e.g. Q1=Q2, this allows, for example, a singlesyringe pump to be used for Q1 and Q2). Then, in this case, Cp,r=2Cp,0, and Q3=2Q1.

[0147] 3-port data on infusion of Na-Hyaluronate (HA) with 100mM NaCl. HA is a biopolyelectrolyte, a polyanion, found in extracellular matrix, aqueous humor, and other biological processes. At very low electrolyte concentration the charges on the linear HA chain are unshielded and the random coil polymer swells due to electrostatic repulsion. ThisAttorney Docket No.11656-005WO1 decreases the light scattering intensity, since the osmotic compressibility is very low due to the strong interchain repulsion. As an electrolyte (NaCl in this example) is added the charges on the chain are electrostatically shielded and the coil contracts, and the interchain repulsions diminish, leading to a greater osmotic compressibility and smaller A2, and hence higher light scattering intensity.

[0148] Figure 16 shows the result of infusion of 100mM NaCl aqueous solution into 1ml of 0.20 mg / ml HA in the cuvette, initially in pure water. The 100mM NaCl solution in a syringe, and a solution of HA in pure water at 0.40 mg / ml in a separate syringe were infused into the cuvette at 0.002 ml / s, under constant stirring from the non-contact stir device, using a dual-head Chemyx Nexus 6000 syringe pump, while fluid was withdrawn from the cuvette at 0.004 ml / s by a second Nexus syringe pump, thus keeping the concentration of HA constant in the cuvette at 0.2 mg / ml, while maintaining the cuvette volume at 1ml. The data show I(t) / I0, where I0 is the initial scattering intensity at [NaCl]=0. The data show the details of how the NaCl reduces the interchain interactions respond to [NaCl], increasing I(t) / I0 by over a factor of 3.5x from 0 to 100mM NaCl.

[0149] Programmed titration paths with multiple access ports. The foregoing demonstration of multiple access ports used constant flow rates for the pumps. It is possible, furthermore, to follow controlled composition paths when using two or more titrants, introduced into the receptacle with programmable flow rate pumps. For example, the Chemyx Nexus 60004-channel pump allows programmable flow rates.

[0150] For N access ports feeding into the receptacle, maintained at constant volume, V0, Z where CY(t) is the variable flowfrom reservoir j, and CVWJis the extraction rate from the receptacle, which can go through one or more flow-equipped detectors, and finally into waste. Let CR,jbe the concentration of solute j in reservoir j, and mj(t) be the mass of solute j in the receptacle at time t. Then, for each component j, the mass of mj in the cuvette is given by >?Y = −?Yand the concentration of soluteAttorney Docket No.11656-005WO1 ^^ ^?^@^ Y@ =EFso that >^Y= ^YCVWJ>@ EF−EFThe time-dependent form of the desired ^Y^@^ in the receptacle.

[0151] For 1 and 2 are to be added to the receptacle, such that1 increases linearly from 0 to a specified maximum concentration C1,max and component 2 decreases from C2,max to 0 linearly. Then ^\^@^ = ]@^\,L^_, 0 < @ ≤ 1 / ]where 1 / a is the desired duration of the infusion period, and ^^^@^ = ^1 − ]@^^^,L^_, 0 < @ ≤ 1 / ]^so thatC\^@^ =^ ^]EF + ]@CVWJ^This gives the expression for the linear increase of C1(t) inthe receptacle, while >^^ ^= −]^ A,^C^^@^^^CVWJ^,L^_ =−and soC^^@^ =^^,L^_^ ^−]EF + ^1 − ]@^CVWJ^A,^This gives the expression for a linear decrease in Q2(t) to achieve the linear decrease in C2(t) in the receptacle. With the condition that Q1(t)+Q2(t)=Qout(t) Qout(t) is determined. The simplest way to ensure the flow balance is to use the method of placing the liquid withdrawal tube at a fixed height in the receptacle. In this case the pump for Qout(t) does not need to be programmed.

[0152] Use of a dual access port device in a continuous flow system. By holding the cuvette volume constant, while adding an external liquid continuously to dilute the polymer or colloid solution initially present, and removing cuvette liquid at an equal rate a continuous record of the polymer or colloid concentration behavior can be obtained. This canAttorney Docket No.11656-005WO1 enable continuous, automatic records in i)-vi), instead of the discrete additions and discrete data points. The cuvette volume can be held constant by matching the pump furnishing the inlet fluid to the pump extracting the cuvette fluid. Another option is to provide a withdrawal tube connected to the withdrawal pump, which can be fixed at a desired height within the cuvette, thus automatically ensuring constant cuvette volume.

[0153] In this case the concentration of polymer, Cp(t) in the cuvette is computed as a function of cuvette feed time by ^"P ^^@^ = ^FeNOJwhere C0is the initial concentration of at fixed volume V0, and a flowrate of Q (volume / second).

[0154] Figure 17 shows the fractional polymer concentration Cp(t) / C0for the following parameters: V0=1ml (labelled Vcuv), C0=0.010 g / ml, Q=0.001 ml / s, volume in reservoir, Vr, is 20ml. It is noted that this method allows spectroscopic measurements over many orders of magnitude in concentration. Because of the continuous non-contact stir there is no need to remove the cuvette for agitation, as is frequently the case when adding discrete aliquots.

[0155] Dissolution kinetics of dry materials. It is frequently desired to know when a dry material introduced into a solution is fully dissolved or fully suspended. Often times, dissolution times are merely assumed, and used as a protocol; e.g. stirring overnight at a given temperature and RPM is a commonly used procedure. However, the dissolution may occur considerably more quickly, and if this is monitored, then significant time can be saved in preparing macromolecular, colloid, and other solutions.

[0156] The stirring device, used alone in a spectroscopic instrument, allows the dissolution to be monitored. Figure 18A shows the light scattering intensity during dissolution of dried gelatin crystals when introduced into water at 40oC, non-contact stirred at 500RPM, at a concentration of 1 mg / ml. The dissolution is complete by 15 minutes, seen by the leveling off of the light scattering intensity.

[0157] Figure 18B shows the weight average molar mass, Mw, versus time while bovine serum albumen (BSA) is non-contact stirred at 500 RPM at T=25oC, at a concentration of 5 mg / ml, into a 5mM PBS buffer at pH=7.4. In contrast to Figure 18A the BSA quickly dissolves into small clusters of protein (~3 BSA molecules, on weight-average), and then drops and converges close to its known Mw of 65,000 g / mol. What appear to be ‘noisy’ points are actually larger clusters of BSA that dissolve more slowly than the majorAttorney Docket No.11656-005WO1 mass portion of the population. The height and density of the ‘cluster signals’ are seen to diminish in time as these clusters also dissolve.

[0158] The devices, systems, and methods of the appended claims are not limited in scope by the specific devices, systems, and methods described herein, which are intended as illustrations of a few aspects of the claims. Any devices, systems, and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the devices, systems, and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compounds, components, compositions, and method steps disclosed herein are specifically described, other combinations of the compounds, components, compositions, and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

[0159] The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than where noted, all numbers expressing geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.

[0160] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Claims

Attorney Docket No.11656-005WO1 What is claimed:

1. A device (100) comprising a motor (102) coupled integrally to a drive shaft (104) and impeller (106), wherein the drive shaft and the impeller are insertable within a receptacle (108) such that the impeller can deliver non-contact stirring or reciprocal motion to a fluid (110) present within the receptacle when driven by the motor.

2. The device of claim 1, wherein the motor comprises a stepper motor electric motor, a non-stepper electric motor, an oscillating motor, or a reciprocating motor.

3. The device of any of claims 1-2, wherein the device further comprises a cap assembly (112) comprising a body portion (114) insertable within an opening of the receptacle.

4. The device of claim 3, wherein the motor is mounted on the cap assembly.

5. The device of any of claims 3-4, wherein the drive shaft extends from the motor through the body portion and into the receptacle when the body portion is inserted within the opening of the receptacle.

6. The device of any one of claims 1-5, wherein the drive shaft is detachably coupled to the motor.

7. The device of any one of claims 3-6, further comprising a mounting post (118) extending from a bottom of the body portion, wherein the mounting post is configured for attachment of a membrane.

8. The device of claim 7, wherein the mounting post is configured for hermetically sealed attachment of a membrane 9. The device of any one of claims 1-8, where the device is positionable within an instrument that performs a spectroscopic measurement, either continuously or at intervals.

10. The device of claim 9, wherein the spectroscopic measurement is chosen from static and dynamic light scattering, UV / visible absorption, infra-red absorption, fluorescence,Attorney Docket No.11656-005WO1 Raman scattering, circular dichroism, circular birefringence, polarimeter, dielectric spectrometer, and microwave absorption.

11. The device of any one of claims 1-10, wherein the fluid contains a biologic drug in molecular, macromolecular, or colloidal form.

12. The device of any one of claims 3-11, wherein the cap assembly allows spectroscopic monitoring of membrane mediated processes.

13. The device of claim 12, wherein the membrane mediated process comprises membrane dialysis.

14. The device of any one of claims 1-13, wherein the device is used in conjunction with an electrode assembly (120).

15. The device of claim 14, wherein the electrode assembly resides independently of the device in the receptacle.

16. The device of claim 14, wherein the electrode assembly is integral to the device and resides inside the receptacle.

17. The device of claim 14, wherein the electrode assembly is external to the receptacle.

18. The device of any one of claims 1-17, further comprising one or more access ports (116) that allow solids, liquids, gases, or a combination thereof to be added to and / or removed from the receptacle.

19. The device of claim 18, wherein the one or more access ports have a cross-sectional area of from 1% to 20% of a cross-sectional area of an opening of the receptacle.

20. The device of any of claims 1-19, wherein the receptacle comprises a spectroscopic cuvette.

21. The device of claim 20, wherein the cuvette has an optical path length of 1 cm or less.Attorney Docket No.11656-005WO1 22. The device of any one of claims 20-21, wherein the receptacle comprises a quartz cuvette, a borosilicate cuvette, or a plastic cuvette.

23. The device of any one of claims 1-22, wherein the receptacle is optically transparent within the UV and / or visible region.

24. The use of the device of any of claims 1-23 to stir a fluid present within a receptacle.

25. A method comprising adding a sample comprising an analyte to a receptacle; inserting the device of any one of claims 1-24 within the receptacle such that the impeller can deliver non-contact stirring or reciprocal motion to the sample present within the receptacle when driven by the motor; and stirring the sample with the device.

26. The method of claim 25, wherein the method further comprises performing one or more spectroscopic measurements of the sample.

27. The method of any one of claims 25-26, wherein the device further comprises one or more access ports that allow solids, liquids, gases, or a combination thereof to be added to and / or removed from the receptacle; and wherein the method further comprises adding a substance to the receptacle or removing a substance from the receptacle via the one or more access ports.

28. The method of claim 27, wherein the substance is added or removed via the one or more access ports at a continuous rate.

29. The method of claim 27, wherein the substance is added or removed via the one or more access ports in one or more aliquots.

30. The method of any one of claims 27-29, wherein the method further comprises performing one or more spectroscopic measurements of the sample prior to addition of the substance or the removal of the substance from the receptacle.Attorney Docket No.11656-005WO1 31. The method of any one of claims 27-30, wherein the method further comprises performing one or more spectroscopic measurements of the sample during and / or after addition of the substance or the removal of the substance from the receptacle.

32. The method of any one of claims 27-31, wherein the method comprises a spectrophotometric titration.

33. The method of any one of claims 27-31, wherein the method comprises adding a substance to the receptacle via the one or more access ports, stirring the sample with the device, and performing one or more spectroscopic measurements of the sample to observe a response to the addition of the substance.

34. The method of any one of claims 27-33, wherein a solvent is flowed through a first access port into the receptacle containing the sample having an analyte therein at a first concentration; and wherein fluid is removed from the receptacle via a second access port, optionally at the same rate, thereby continuously reducing the first concentration of the analyte.

35. The method of claim 34, further comprising spectroscopically monitoring the sample to assess one or more concentration-dependent properties of the analyte.

36. The method of claim 35, wherein the one or more concentration-dependent properties of the analyte can comprise weight average molar mass, virial coefficients, electrical conductivity, UV / visible absorption, fluorescence, or a combination thereof.

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