The Zetasizer Helix delivers structural information at the molecular level, providing mechanistic insight into the formation of oligomers, unfolding, and aggregation processes of biopharmaceuticals.
The system retains all of the functionality of the Zetasizer Nano ZSP and adds Raman spectroscopy to determine secondary and tertiary structure of proteins and protein-based biotherapeutics. This wealth of characterization data provided by a single instrument platform provides the understanding to enable more efficient drug development processes, shortening time to market, and increasing drug product quality. The exceptional performance also enables the measurement of the molecular weight and second virial coefficient, A2, of macromolecules and kD, the DLS interaction parameter.
Information can be derived from non-experts through powerful software – where trends of a pre-determined set of parameters are graphed and fitted with no expertise required.
To keep from sacrificing analytical power though, an advanced processing mode provides a full suite of tools – from data pre-processing to multivariate analysis. The use of the instrument is therefore enabled for applications method development.
- Study formulations without or with minimal dilution
- Understand mechanisms of aggregation and oligomerization
- Identify Critical to Quality product attributes
- “Expertise Optional” data analysis software provides trend results without making expert level decisions
- Advance data analysis tools include a full suite of spectroscopic analysis tools, from pre-treatment to multivariate analysis.
- Temperature ramp data gives insights into product stability and mechanisms of aggregation
- Isothermal incubation data enables kinetics and reversibility to be investigated
- Sample series allows the cause of batch-to-batch variation to be investigated
- In-depth analysis of secondary and tertiary protein structure
How it works
By combining Raman spectroscopy (to characterize secondary and tertiary protein structure) with dynamic light scattering (to characterize particle size) the Zetasizer Helix can differentiate between the following mechanisms:
- Formation of oligomers at the same time as 2e and/or 3e structural change
- Formation of oligomers with no change to 2e and/or 3e structure
- Protein unfolding without resulting aggregation
- Aggregation as a result of unfolding/structural change
- Aggregation without unfolding/structural change
Because of richness of detail provided by the Raman spectra, it is possible to correlate the above processes (pertaining to structure and aggregation) with specific protein moieties and their environments. This information can be provided on a single product, or used to compare these properties between different products and/or batches
The system has significant flexibility in enabling a variety of sample stressors to be applied, and the effects studied.
Data collection options and results obtained:
- Kinetics / Isothermal Incubation / Temperature Jump
- Determine kinetics of oligomerization and/or aggregation events
- Investigate reversibility of size and structural changes during these processes
- Thermodynamics / Temperature Ramp
- Determination of thermodynamic properties
- Tonset, Tmelt
- Enthalphy/coopertivity of transitions
- Compare relative stability of samples
- Mechanistic understanding of oligomerization and aggregation events
- Sample Series
- Evaluate impact of a perturbation across a range of samples
- Compare stability and molecular structure of different batches, or as a function of stability testing,
- Perturbation other than temperature (pH, denaturant, etc.)
Data collection and data analysis are handled separately to present a clearly delineated work flow. Data collection is controlled by ZSHelixAcquire, which provides a “wizard-type” interface, guiding the user through experimental setup. This interface is launched in the Zetasizer software. Frequently used settings are saved as “experiments” (methods), and can be loaded and reused for ease-of-use and guaranteed reproducibility of experimental conditions.
ZS HelixAnalyze is the powerful yet easy to use data analysis platform. The default “Expertise Optional” mode combines data pre-treatment, processing and trend analysis into a single automated sequence that requires no “Expert” decision to complete. To keep from sacrificing analytical power though, an advanced processing mode provides a full suite of spectroscopic tools – from data pre-processing to multivariate analysis. The use of the instrument for novel and unanticipated uses is therefore not limited, enabling applications method development, and ensuring the usefulness of the system for as yet unidentified applications.
- DLS light source:
- He-Ne laser 633nm, Max 10mW
- Raman light source:
- Diode laser 785nm, Max 280mW at the sample
- Laser safety:
- Class 1
- < 100VA (DLS)
<300VA, typical operating output ~135VA (Raman)
- Dimensions (W, D, H):
- 320 x 600 x 375mm (Zetasizer)
580 x 450 x 200mm (Raman)
- 19kg + sidecar (Zetasizer)
- Structural and chemical analysis:
- Raman spectroscopy
- Raman spectrometer:
- Kaiser Optical Systems Inc. RamanRxn1
- Spectral performance:
- 150 cm-1 to 1850 cm-1, 6 cm-1 resolution
- Laser spot size at sample:
- <300 µm
- Protein secondary and tertiary structural markers:
Backbone – amount of α-helix, ß-sheet, and random coil content
Tyrosine – hydrogen bonding extent, hydrophilic or hydrophobic environment
Tryptophan – indole ring dihedral angle to backbone, extent of cation-π interaction, if indole ring is buried or exposed, hydrophilic or hydrophobic environment
Disulfide – differentiate conformers, loss of covalent bond
- Data collection modes:
- Isothermal Incubation, Temperature Ramp, Temperature Jump, Sample/Formulation Series
The Zetasizer Nano range is protected by the following patents: Non-Invasive Back Scatter (NIBS) EP884580, US6016195, JP11051843 High and Low Frequency Electrophoreses (M3) EP1154266, US7217350, JP04727064 Light Scattering Measurements using Simultaneous Detection EP2235501, CN102066901, JP2011523451, US20090251696 Surface Potential Determination in a Dip Cell WO2012172330
The Zetasizer Helix is the subject of patent application WO2013027034 A1
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