Adjuvant Research Team

Instruments

• Sunshine LNP Process Developer from Unchained Labs
• Repligen KrosFlo KR2i Tangential Flow Filtration (TFF) system
• LV1 imcrofluidizer homogenizer from Microfluidics
• Fluigent Flow EZ air pressure-based flow system with single-emulsion chip
• Avanti mini extruder with temperature block

Sunshine LNP Process Developer from Unchained Labs

The Sunshine is part of a larger, scalable nanoprecipitation processing platform developed by Unchained Labs and uses precise microfluidics engineering to tightly control nanoprecipitation processes to form and load nanoparticle formulations.

Find more information about the Sunshine on the Unchained Labs website.

Repligen KrosFlo KR2i Tangential Flow Filtration (TFF)

Filtration is typically performed via a flow-through method by passing samples through a membrane. Tangential flow filtration (TFF) instead flows the sample parallel, past a permeable membrane and applies a tangential flow across the membrane using via pressure, reducing membrane fouling and allowing highly scalable volumes of sample to be processed using a much smaller membrane surface areas than would be needed in flow-through filtration.

TFF is a method used for separating, purifying, and concentrating samples and the KrosFlo KR2i allows us to automate this process via a program of alternating diafiltration and concentration steps. Where diafiltration would normally takes many days and ultimately dilute a sample, TFF allows us filter quickly and further concentrate a sample as we wish without spending valuable labor time actively monitoring the instrument.

Instruments

• 2x Covaris S2 focused ultrasonicators with recirculating heater/cooler controllers
• 1x Covaris S2 focused ultrasonicator with thermoCUBE solid-state heating/cooling controller
• 3x Bath sonicators
• 1x Probe sonicator

Focused Ultrasonication vs Bath or Probe Sonication

Covaris S2 instruments use the same sonic energy used in traditional bath and probe sonicators, but focus this energy into a single point in a temperature-controlled, closed vial. Bath sonicators allow samples to be processed in a closed-system, but disperse their energy widely, resulting in weaker processing power. Probe sonicators produce far stronger processing power, but must be inserted directly into a sample, leading to metal contaminants leaching into the formulation. Both methods require careful, manual control of the sample temperature.

Covaris instruments overcome the limitations of both of these methods. Focused ultrasonic energy produces cavitations within the sample, allowing for probe sonicator levels of processing power, but within a closed, sterile system like a bath sonicator. Additionally, the Covaris instruments include temperature-control systems to precisely and efficiently maintain sample temperature during long-processing runs. The systems we have in our lab are setup to operate from a range of about 1 to 25 mL volumes per vial.

Our labs has a variety of liquid mixer-type homogenizers that cover the range from small to large scale volumes. Liquid mixer-type high-shear homogenizers utilize shear-stress to process samples into smaller sizes and increase their homogeneity. These instruments are similar to a blender in a kitchen but with far more precision and power.

Instruments

• Silverson L5M-A homogenizer mixer (large scale)
• Ika T25 Easy Clean Control Ultra-Turrax disperser (small to medium scale)
• Heidolph Silent Crusher S homogenizer (small scale)

Instruments

• Vacuum Concentrators
  • Centrifugal vacuum concentrator, SpeedVac SPD140DDA, Thermo Scientific with Savant RVT5105 refrigerated vapor trap
  • Multi-sample rotary evaporator, Multivapor P-12, Buchi
  • Rotary evaporators
• Stability
  • Forma Environmental Chamber (UV light, moisture, heat)
• Ovens
  • Thermal/UV Stability chamber
  • Depyrogenation oven
  • Benchtop Muffle Furnace, Thermolyne Furnace FB1415M, Thermo Scientific (programmable, 100° - 1100°C
  • Benchtop oven (30° - 300°C)
• Centrifuges
  • Ultracentrifuge, Sorvall WX 80 with Surespin 630 rotor (up to 30000 rpm, 33mL)
  • Benchtop centrifuges
• Other
  • 2x BioChemGard Hoods (sterile, vented chemical work)
  • Syringe pumps
  • Peristaltic pumps

LCMS

• Thermo Scientific Exploris480 Q-Orbitrap nLC/MS with VanquishNeo nanoLC
  
  The Exploris480 is a proteomics powerhouse capable of quantifying thousands of proteins in a single run. Equipped with the second generation FAIMS Pro Duo, this instrument has incredible sensitivity down to the single cell level. The BioPharma option also allows the Exploris480 to perform top-down protein analysis for characterization of PTMs or protein-drug conjugates. Utilizing DDA, DIA, and hybrid-DIA acquisition, methods can be tailored for nearly any biologic question, allowing investigators to see global cellular changes in as little as a 9 minute run. With a maximum resolution of 480,000, the Exploris480 excels at metabolomic analysis as well. Simultaneous quantitation and discovery (SQUAD) methods allow for targeted quantitation of specific metabolites while also collecting high resolution all ion spectra, so researchers never miss a potential molecule of interest.

MALDI-TOF

• Bruker Microflex MALDI-TOF MS

UPLCs

• Vanquish Flex UHPLC with binary pump, Corona Veo CAD and diode array detector, max pressure ~700 bar, Chromeleon 7.3 software
• Vanquish Flex UPLC with binary pump, CAD, and variable wavelength detector (4 channels, no 3D scan), max pressure ~1000 bar, Chromeleon 7.3 software
• Waters Acquity Arc with quaternary pump, photo diode array detector, fluorescence detector, max pressure ~650 bar, Empower 3 software

HPLCs

• 2x Waters Alliance with quaternary pump and photo diode array detector, max pressure ~275 bar, Empower 3 software
• Agilent 1200 with binary pump and diode array detector, max pressure ~400 bar, Agilent Openlab software
• Agilent 1100 with quaternary pump, diode array detector, max pressure ~275 bar, Agilent Openlab software

Asymmetrical-flow field flow fractionation (AF4) is a an alternative method of separating samples via particle size to size-exclusion chromatography (SEC). Rather than using a column for separation, AF4 uses a permeable membrane and tunable asymmetric flow patterns to separate and focus samples within the flow path. This method is gentler on samples than SEC and typically allows for superior control of analyte elution over SEC while also providing the opportunity to utilize AF4 theory for particle size analysis. These systems are highly flexible and powerful in their capabilities for analyzing nanoparticles, polymers, and proteins.

Instruments

• Postnova AF2000 Multiflow FFF Universal Separator
  • Analytical flow column
  • Refractive Index (RI) detector
  • UV-VIS (2 channel)
  • Multi-Angle Light Scattering (MALS), 21 angles
  • Dynamic Light Scattering (DLS), Malvern Zetasizer ZS
  • Autosampler
  • Manual injector
  • NovaAnalysis software

Instruments

• 2x Malvern Zetasizer ZS ZEN3600 (13° or 173°)
  
  • UV-Micro-volume cuvettes, ZEN0040 (50 - 850 uL)
  • Quartz Flow Cell, ZEN0023
  • Folded capillary cells with gold electrodes, DTS1070 (750uL)
  • Dip Cell with palladium electrodes, ZEN1002 (600 - 750uL)
• Postnova MALS (21 angles, 7° to 164° simultaneously)

Analysis Modes

• Dynamic Light Scattering (DLS): Used for determination of Hydrodynamic Size and distribution. Can be run in both batch or flow mode.
• Multi-Angle Light Scattering (MALS): Can be used to find absolute molecular weight, but also provides Gyration Size
• Electrophoretic Light Scattering (ELS): Provides charge information such as Zeta Potential and distribution.
• Static Light Scattering (SLS): Used to estimate protein molecular mass. Must use a quartz cuvette.

Instruments

• Vapor Pressure Osmometer, Vapro 5600, EliTechGroup
• Circular Dichroism (CD) Spectrometer, Jasco J-1500 CD
• Portable Endotoxin Testing system, Endosafe nexgen-PTS, Charles River (LAL, rCR, beta-glucan)
• Microvolume Spectrometer/Fluorometer, Denovix DS-11 FX+
• Analytical balances
• pH electrodes, small volume
• Conductivity meter