U6. Biomaterial Processing and Nanostructuring Unit – Equipment

U6-E16. Freeze Dryer

Freeze drying (also known as lyophilization) is a water (or other solvents) removal process typically used to preserve materials, to extend their shelf life or reduce its weight. Freeze drying works by freezing the material, then reducing the pressure and adding heat to allow the frozen water in the material to change directly to a vapor (sublimation).

Freeze drying occurs in three phases:

             1-Freezing

Freezing can be done in a freezer, a chilled bath (shell freezer) or on a shelf in the freeze dryer. Cooling the material below its triple point ensures that sublimation, rather than melting, will occur. This preserves its physical form.

              2-Primary Drying

Freeze drying’s second phase is primary drying (sublimation), in which the pressure is lowered and heat is added to the material in order for the water to sublimate. About 95% of the water in the material is removed in this phase. Primary drying can be a slow process.

             3-Secondary Drying

Freeze drying’s final phase is secondary drying (adsorption), during which the ionically-bound water molecules are removed. Most materials can be dried to 1-5% residual moisture.

Technical Specifications:

• Vacuum pump: 7 m³/h
• Condenser min. Temperature: – 80º C

Aplications:

Solvent removal is typically used to preserve materials, to extend their shelf life or reduce its weight.

U6-E05. Tangential flow filtration system

Tangential flow filtration (TFF)  is a process of separation widely used in bio-pharmaceutical and food industries. It is different from other filtration systems in that the fluid is passed parallel to the filter, rather than being pushed through a membrane perpendicularly which can clog the filter media. This method is preferred for its continuous filtration and reproducible performance. The particles that pass through the membrane, the permeate, are put off to the side, while the rest, the retentate, is recycled back to the feed.

 Technical Specifications:

• Volume: 10 ml
• Filtration volume rate: 0.01 to 2300 ml/min

Aplications: Tangential flow filtration is used in the following processes:

Concentration: Increases the concentration of a solution by removing fluids while keeping the solute molecules. This process is done by selecting a filter significantly smaller than the solute molecules to allow for a higher retention of solute molecules.

 Diafiltration:  The separation of small and large particles, leaving the smaller particles behind without altering the overall concentration.

U6-E08. Mastersizer 2000

Mastersizer 2000 – Malvern

Description:

The Mastersizer 2000 uses the laser diffraction technique to measure the size and particle size distribution of materials. This is carried out by measuring the intensity of light scattered when a laser beam passes through a sample of dispersed particles. The detectors measure light intensity for red and blue light. The data is analyzed to calculate the size of the particles that created the scattering pattern.

The equipment contains two sample dispersion units:
– Wet: for samples in solution, which can be applicable to different solvents.
– Dry: for solid samples in powder form, which are dispersed by a current of air.

Technical specifications:

• Particle size: 10 nm – 3.5 mm
• Measurement principle: Mie and Fraunhofer dispersion
• Light source:
– Red: He-Ne (632.8 nm)
– Blue: LED (470 nm)
• Optical Alignment: Automatic rapid alignment system with darkfield optical reticle and multi-element alignment detector
• Dispersion units:
– Dry route
– Wet route
• Operating temperature: 5º to 40º C

Applications:

• Molecular and particle size distribution analysis.
• Powder fluidity analysis
• Particle compression density analysis
• Stability analysis in suspensions and emulsions
• Analysis of the dissolution rate of materials
• Determination of the reaction rate in solid systems

U6-E10. Nanosight NS-300 for Nanoparticle Tracking Analysis by fluorescence mode.

NanoSight NS300 – Malvern

Description:

The NanoSight NS300 uses nanoparticle tracking analysis (NTA) technology, which uses the properties of light scattering and Brownian motion to obtain measurements of concentration and size distribution of particles in liquid suspension.
A laser beam passes through the sample chamber, and suspended particles in the path of this beam scatter the light in such a way that they can be easily seen through a 20x microscope on which a camera is mounted. The camera runs at 30 frames per second and captures a video of the particles in their natural Brownian motion. The software tracks the particles individually and, using the Stokes-Einstein equation, calculates the hydrodynamic diameter of each of them.

Technical specifications:

• Size range: 10 – 1000 nm
• Concentration range: 106 – 109 particles/ml
• Temperature control range: 5º C below ambient to 50º C
• Minimum sample volume: 250 µl
• Camera: SCMOS
• Focus: Computer controlled motorized focus
• Fluorescence: 6-place motorized filter wheel, with filter options
• Wavelength:
– Blue: 488nm
– Green: 532nm

Applications:

• Molecular and particle size distribution analysis.
• Concentration measurement.

Update of the Nanosight system

1. The Green laser module for the NanoSight NS300 equipment.

The NS300 allows analysis of the size distributionand concentration of all types of nanoparticles from 0.01 – 1 µm in diameter. This new laser module will allow NTA to measure a range of fluorescent particles, avoiding interferences during the measurements due to sample (auto)fluorescence and absorption.  This is done by detecting the fluorescence signal, which is emitted naturally by particles or as a result of fluorescence labeling or tagging.

Financed by the project FICTS-1420-27

         2.  Nanosight NS300 Violet Laser Module 405nm

Description: The NanoSight NS300 instrument provides a reproducible and easy-to-use platform for nanoparticle characterization. The NS300 allows for quick and automatic analysis of size distribution and concentration of all sorts of nanoparticles, ranging from 10 to 1000 nm in diameter, depending on instrument configuration and sample type. Thanks to the possibility of adding interchangeable laser modules and the introduction of a 6-position motorized filter wheel, analysis of different fluorophores can be performed. The sample temperature is fully programmable through the nanoparticle tracking analysis (NTA) software.

Technical Specifications:

• Size Range: 10 nm-1000 nm
• Concentration Range: 10^6-10^9 particles/mL
• Minimum Sample Volume: 250 µL
• Laser Type: 405 nm violet laser
• Fluorescence Filter: 430 nm
• Temperature Range: 5 ºC below ambient temperature up to 70 ºC

Applications:  The equipment can be used in various applications and to characterize different types of particles, such as: Extracellular vesicles, Viruses and vaccines, Drug delivery, Biotherapeutics, Colloids and Nanomaterials, Ultrafine bubbles.

Financed by the project ICT2021-006987

U6-E17. Surface Plasmon Resonance (SPR) system

SR7000 DC Reichert

Description: Dual Channel Modular platform, outstanding value. This Surface Plasmon Resonance (SPR) system generates high-quality data for kinetics, affinity, thermodynamics, and concentration. The SR7000DC SPR System provides SPR technology for label-free biomolecular interaction analysis.

Reichert’s SR7000DC SPR System is an innovative label-free detection system offering unparalleled flexibility and remarkable sensitivity for biomolecular and biochemical analysis. Designed for breakthrough discoveries, it is component-based and cost-effective; upgrade or add instruments at any time. The system is low maintenance, using off-the-shelf fluidics.

 Technical specifications:

Measurement Channels: Two

Sample Loading: Autosampler. Up to 768 samples

Injection Volume: 1 uL to 4,500 uL (depends on installed loop volume)

Buffer Degasser: Built-in

Temperature Range: 10 °C below ambient to 70 °C

Sample Storage: 4 °C or ambient temperature

21 CFR Part 11: Compliant

Measurement sensitivity

Baseline Noise: 0.05 µRIU (RMS)

Baseline Drift: < 0.1 µRIU/min

Minimum Molecular Weight Detection: No lower limit for organic molecules

Typical kinetic and equilibrium constant ranges

Association Rate Constant: 10³ to 10⁸ M^-1 sec^-1

Dissociation Rate Constant: 10^-1 to 10^-6 sec^-1

Equilibrium Dissociation Constant: 10^-3 to 10^-12 M

 Applications: SPR is typically used in academic and industry settings to advance research in a range of areas, including:

• Antibody development.
• Drug development and discovery.
• Concentration analysis.
• Gene regulation.
• Nanoparticles/Nanomaterials.
• Pharmaceutical research.
• Studying protein structure and function determination.
• Systems biology.
• Thermodynamics analysis (ΔH & ΔS).

Whether you’re studying Alzheimer’s disease, developing new vaccines or focused on other areas of protein research, SPR can help you quickly determine whether proteins interact, and which ones are worth studying more.

U6-E18. DOE

Design of experiments (DOE) software

Description: Software platform and assistance for performing Design of Experiments (DOE), a systematic and statistical approach to planning, conducting, and analyzing experiments or tests. It involves strategically manipulating variables to gather relevant and reliable information while minimizing resources and time.

Applications: DOE helps researchers and engineers understand the relationships between factors, identify optimal conditions, and improve processes by optimizing performance and reducing variability. Applicable in most fields of production and research.

U6-E15. Microplate reader

U6-E11. Turbiscan Lab

Turbiscan Lab

Description: Turbiscan is used to detect at an early stage all kinds of destabilisation such as coalescence, flocculation, creaming, sedimentation, etc… Various products such as emulsions, suspensions or foams can be studied from low to high concentrations without any sample preparation or dilution. Stability kinetics and index are measured for an efficient sample analysis and comparison.

Applications: Stability studies

U6-E03. Four Glove Dispensing isolator for expermiments with highly active and cytotoxic compounds

Four Glove Dispensing isolator for expermiments with highly active and cytotoxic compounds.

Description: The four-glove isolator is a safe chamber, with controlled environment for the weighing and handling of potent drug compounds.

Technical specifications: The Isolator has been designed to offer containment to a working OEL (Occupational Exposure Limit) of less than 0.1Jgrams/m³ time weighted average over an 8hr operational period (TWA).

The Isolator is designed to offer an internal air cleanliness of Class 8 as described in ISO 14644-7 and the test pass criteria is a leakage rate of ≤0.25% vol/hr and is in line with Class 2 classification of ISO 14644-7.

• Internal Environment: – Main Chamber: -100Pa +/- 10%.
• Air Changes per hour: Nominal 20 during normal operating conditions giving ISO 8.
• Glove Breach Velocity: >0.5m/sec.
• Exhaust System: Max air flow 108m3/hr.
• Inlet HEPA Filter: H14 Canister (safe-change when changed into Isolator). 99.995% efficient @ 3Jm particle size.
• Exhaust HEPA Filter: 1st Stage – H14 HEPA & 2nd Stage – H14 HEPA mounted within Push-Push Safe Change housing 99.995% efficient @ 3Jm particle size.

Applications:

Pharmaceutical Manufacturing: In pharmaceutical manufacturing, especially for the production of potent drugs or active pharmaceutical ingredients (APIs), a 4-glove isolator can be used for containment. It helps in preventing cross-contamination and ensures the safety of both the product and the operator.

Biotechnology Research: In research laboratories working with hazardous biological materials such as viruses, bacteria, or genetically modified organisms (GMOs), a 4-glove isolator provides a secure environment for manipulation and experimentation. It minimizes the risk of exposure to harmful agents.

Sterile Compounding: In compounding pharmacies and hospital pharmacies, a 4-glove isolator can be utilized for sterile compounding of medications. It helps in maintaining aseptic conditions, preventing contamination, and ensuring the safety and efficacy of compounded drugs.

U6-E14. Microcalorimeter for measurements under Isothermal Titration Calorimetry (ITC)

MicroCal VP-ITC – Malvern

Description:

Isothermal titration calorimetry (ITC) measures the binding affinity and thermodynamics between two biomolecules. When substances bond, heat is released or absorbed. Measuring this heat change in an ITC experiment allows precise determination of the binding constants (KD), stoichiometry (n), enthalpy (ΔH) and entropy (ΔS), providing a complete thermodynamic profile of the molecular interaction in a single experiment.
The MicroCal VP-ITC system is a powerful, sensitive and easy-to-use calorimeter that offers the possibility of investigating any biomolecular interaction. This rapid, convenient, label-free, and solution-based technique provides the information needed to characterize the molecular interactions of proteins, antibodies, nucleic acids, and other biomolecules.

Technical specifications:

• Cell type: Non-reactive Hastelloy
• Sample volume 2 mL
• Volume per cell: 1.4 mL
• Volume injected: 0.3 mL
• Mixing speed: user selectable
• Response time: user selectable
• Working temperature: 2º to 80º C

Applications:

• Characterization of molecular interactions of proteins, antibodies, nucleic acids and other biomolecules.
• Kinetic enzyme.
• Evaluation of the effect of molecular structural changes on binding mechanisms.
• Evaluation of biological activity.