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Physiological processes are regulated by molecular mechanisms involving protein-protein and protein receptor-ligand interactions. With the era of evolving biologics such as monoclonal antibodies or biosimilars, biophysical characterization has gained an important role in the development of protein therapeutics. The main analytical methods of biophysical characterization are used to measure binding affinity and kinetics, binding thermodynamics, aggregation and sizing, stability and higher order conformations.

DKSH’s Center of Excellence hosts the most comprehensive selection of biophysical characterization tools in the ASEAN region. For all stages of drug discovery and development, biophysical characterization plays a vital role in determining the safety and efficacy of candidates. Additionally, our tools allow us to generate stable drug substances and stable environments, two critical steps from early in the discovery phase to fill and finish. We offer an extensive portfolio of tools such as SPR, ITC, switchSENSE, QCM-D, DLS, Flowcam, SEC-MALS, DSC, NTA and NanoFCM.

Our portfolio of tools

MMS is a novel technology for label-free biomolecule analysis of protein aggregation, quantitation, stability, structure and similarity that directly addresses the limitations of current technologies. MMS provides drift-free, background subtracted, high sensitivity measurements of the protein secondary structure to facilitate major improvement in development of biopharmaceuticals.

Application Areas

  • Protein Drugs
  • Biosimilars
  • AAVs
  • Peptides
  • Nucleic Acids

Parameters

  • Aggregation
  • Quantitation
  • Stability
  • Similarity
  • Structure

SPR binding studies are used in screening and development studies and can run in various formats for full characterization of biomolecular interactions. High-throughput screening methods can accelerate the lead selection and optimization process from months to weeks.

Application Areas

  • Biologics development
  • Drug discovery
  • Epitope Mapping/Binning
  • Competition Assays

Parameters

  • Binding kinetics (kon, koff)
  • Steady-state affinity (KD)

ITC is the gold-standard for binding affinity, stoichiometry and thermodynamics measurements. The addition of stoichiometry and thermodynamics have been proven to be necessary in understanding edge-cases in binding data. For example, candidates with similar affinities and kinetics have been known to act differently due to their binding thermodynamics.

Application Areas

  • Thermodynamics analysis
  • Drug discovery
  • Compound and fragment screening
  • Protein-protein interactions

Parameters

  • Steady-state affinity (KD)
  • Binding stoichiometry (n)
  • Binding thermodynamics (∆H, ∆S, ∆G)

The switchSENSE by Dynamic Biosensors is a cutting-edge biomolecular interactions system that uses nucleic-acid nano-cantilevers to measure the binding affinity and kinetics, thermodynamics, and conformational changes all in one measurement. This system is a breakthrough for DNA-binding proteins which may be hard to measure with other ligand-binding assays.

Application Areas

  • RNA, DNA-binding proteins
  • Enzymatic function analysis
  • Ternary complex analysis
  • Sizing and conformational change analysis

Parameters

  • Binding kinetics (kon, koff)
  • Steady-state affinity (KD)
  • Conformational change (% friction change)
  • Binding thermodynamics (∆H, ∆S, ∆G)
  • Nucleic acid enzyme activity (kCAT, KM, kEXO)

QCM-D provides valuable interactions data on biomolecules and surfaces. This tool has uses in characterizing the stability of therapeutic agents in its container surfaces, the interaction between protein and its excipients, and in bio-membranes research.

Application Areas

  • Protein-packaging material compatibility
  • Protein-excipients compatibility
  • Bio-membranes characterization

Parameters

  • Binding kinetics (kon, koff)
  • Steady-state affinity (KD)
  • Film thickness
  • Viscoelasticity

DLS is a popular technique for the characterization of particle size and zeta potential. These key parameters can measure the stability of biomolecules over a period of time as a function of particle size and give insight to the self-aggregation of proteins.

Application Areas

  • Protein quality control
  • Protein aggregate analysis
  • Characterization of API and excipients
  • Pre- and formulation development

Parameters

  • Particle size (rh, PDI)
  • Zeta potential
  • Aggregation Analysis

Flow Imaging Microscopy is an orthogonal imaging technique for the characterization of particle size and aggregation. Specifically designed to generate high-resolution images for particles suspended in fluids, Flowcam enables direct evaluation of the quality and safety of parenteral formulations.

Application Areas

  • Particles characterization in fluids
  • Microencapsulation formulation and QC
  • Characterization of API and excipients

Parameters

  • Shape parameters
  • Morphology parameters
  • Fluorescence-modes

SEC-MALS is a separation and detection method of proteins based on size to determine the molar mass of the proteins. This powerful tool allows for the characterization of molecular properties such as molecular weight and can be additionally used for orthogonal aggregation and higher order structure studies.

Application Areas

  • Polymer research
  • Relating molecular properties to bulk characteristics
  • Degradation and drug delivery rates
  • Aggregation studies
  • Higher order structure studies

Parameters

  • Absolute molecular weight
  • Intrinsic viscosity
  • Concentration

DSC is a highly sensitive biophysical tool used to detect changes in temperature and heat capacity change associated with folding and unfolding of proteins. With the DSC, you can understand the thermodynamic reasons for aggregation and stability and is a strong tool for detecting small batch-to-batch changes, allowing for comparability and consistency and establishing biosimilarity.

Application Areas

  • Structural stability analysis
  • Batch-to-batch comparability
  • Optimization of purification and manufacturing conditions

Parameters

  • Temperature midpoint (TM)
  • Enthalpy (ΔH)
  • Heat capacity change (ΔCp)

NTA is the premier imaging technique for the characterization and analysis of exosomes. The technique can monitor individual particles by measuring the Brownian motion of particles in diffusion, which relates to the particle size. Exosomes play a crucial and developing role as both drugs and drug delivery vehicles.

Size Range

  • 10nm – 1000nm in solution

Advantages

  • Gold standard for exosome analysis
  • Get size and distribution in real time
  • Further differentiation with fluorescence

NanoFCM is the only flow cytometry tool capable of measuring down to the smallest exosome levels (30-1000nm). The ability to generate meaningful biochemical data through flow cytometry makes NanoFCM a great orthogonal tool to the NTA.

Size Range

  • 30nm plus

Advantages

  • Single-exosome capability
  • Liquid biopsy applications with immunostaining

Binding Affinity and Kinetics

Binding affinity and kinetics tell you how strongly two biomolecules interact with each other and the kinetic rates of association and dissociation. Also called biomolecular interactions, affinity and kinetics data build the backbone of protein therapeutics drug discovery by providing information on the interactions between protein to drug and protein to protein. The determination of KD and Kon Koff values are required for IND/CMC programs.

  • Label-free interactions methods such as SPR and ITC give best-in-class quantitative data on binding affinity and kinetics
  • Obtain information in real time
  • High-throughput epitope binning and fragment-based discovery

Instruments

Malvern Panalytical MicroCal ITC range
The "Gold Standard" for measuring binding affinity and stoichiometry

Nicoya OpenSPR Instrument
World’s only benchtop surface plasmon resonance (SPR) instrument

Nicoya Alto Instrument
World's first SPR instrument to integrate digital microfluidics (DMF) with nanotechnology-based biosensors

Aggregation and Sizing

Protein aggregation is the self-association of proteins and commonly tested for in biomedical studies and development of biologics. Aggregation of proteins are the causes of several well-known diseases such as Alzheimer’s disease and aggregation in drugs can lead to a loss in efficacy. Therefore, protein aggregates are characterized based on their size, kinetics, conformation, chemical modification, and morphology in order to optimize and minimize their ability to aggregate.

  • Obtain particle size distribution results in isolation or in formulation
  • Gain key insights into how aggregation forms, how fast it forms, and perform comparability studies.

Instruments

Malvern Panalytical Expert Light Scattering Zetasizer Advance range
The world’s most widely-used systems for nanoparticle, colloid and biomolecular particle sizing and particle charge measurements

Malvern Panalytical multi-detector GPC/SEC system OMNISEC
The world’s most advanced multi-detector GPC/SEC system

Malvern Panalytical MicroCal DSC range
The "Gold Standard" for measuring binding affinity and stoichiometry

Stability and Higher Ordered Structures

Biopharmaceutical stability studies are essential to assess and monitor the pathways to loss in biologic activity, safety or quality. Common factors impacting stability can be aggregation, degradation of proteins, conformational changes, biological activity, impurities, and the degradation of excipients. By understanding the degradation pathways, CPPs and CMAs can be better assessed and controlled. It is critical that all parts of the formulation and their interactions are well-characterized using standardized analytical methods.

  • Gain key insights into the stability of proteins and protein therapeutic formulations
  • Characterize higher ordered structures of proteins to find high conformational integrity candidates
  • Characterize higher ordered structures of protein therapeutic formulations to understand how excipients, impurities and container surfaces can cause conformational change.

Instruments

Ability to measure wide range of native sample concentrations from <0.1 mg/ml to >200 mg/ml

RedShiftBio AQS³pro
AQS³pro is designed to provide five key measurements— aggregation, quantitation, stability, similarity, structure—directly supports biomolecule characterization throughout the biopharmaceutical development pipeline

Constant Climate Chambers
Humidity test chambers with ICH-compliant light source

Malvern Panalytical MicroCal DSC range
The "Gold Standard" for measuring binding affinity and stoichiometry

Malvern Panalytical multi-detector GPC/SEC system OMNISEC
The world’s most advanced multi-detector GPC/SEC system

Malvern Panalytical Expert Light Scattering Zetasizer Advance range
The world’s most widely-used systems for nanoparticle, colloid and biomolecular particle sizing and particle charge measurements

Exosome Characterization

Exosomes (extracellular vesicles) are a key recent development in the biomedical and biopharmaceutical industry. Involved in a wide range of healthy and pathogenic processes—cancer, inflammation, immunity, CNS function, cardiac cell function, to name a few—exosomes are being studied for their role in these basic biological processes as well as for their use as biomarkers and even as tools for targeted delivery of biomolecules such as protein therapeutics.

Related products

Malvern Panalytical NanoSight Range
The Malvern NanoSight range of instruments provides an easy-to-use, reproducible platform for nanoparticle characterization.

Particle Metrix ZetaView Basic PMX-120

Particle Metrix - ZetaView
For the analysis of size, concentration, fluorescence, electrophoretic mobility and sub-populations of individual nanoparticles

NanoFCM Flow Nanoanalyzer
High sensitivity flow cytometry for nanoparticle analysis. The only real flow product in the world with the ability to analyze single nanoparticles.

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