Need of a single biophysical tool to characterize and detect changes in the higher order structure (HOS) of a protein drug.
Measure signals from as small a unit of the protein as possible.
2. Detect all such signals emitted from a single molecule.
3. Separate signals spatially in a manner using some parameter space.
4. Quantitatively record all the signals with the highest precision (and accuracy) possible.
Multivibrator and its types defination and usges.pptx
Strand class of biophysical tools used in biophysical industry
1. ID: 190707
MD HABIBUR RAHMAN
TASIN
2ND YEAR 2ND TERM
Biotechnology and Genetic Engineering Discipline
2. INTRODUCTION
Need of a single biophysical tool to characterize and
detect changes in the higher order structure (HOS) of
a protein drug.
1. Measure signals from as small a unit of the protein as
possible.
2. Detect all such signals emitted from a single molecule.
3. Separate signals spatially in a manner using some parameter
space.
4. Quantitatively record all the signals with the highest
precision (and accuracy) possible.
3. GROUP OF BIOPHYSICAL TOOLS
1.Standard Biophysical Tools
• Most applicable and
dominant used by
biopharmaceutical
companies.
2. Advanced Biophysical Tools
• Used in a limited capacity.
• Generate information with greater
detail and higher resolution.
• Success rate of drug approvals.
• Reduce cost and time.
• Providing detailed picture.
4. THE STANDARD CLASS OF BIOPHYSICAL TOOLS
Tier 1 Biophysical Tools
Low-resolution spectroscopy
tools.
Consisting of UV, fluorescence,
circular dichroism (CD) etc.
Provide the basic secondary
and tertiary structure
fingerprint.
Static Light Scattering/Dynamic
Light Scattering (SLS/DLS) and
particle techniques.
Tier 2 Biophysical Tools
Analytical Ultracentrifugation
(AUC) and Differential
Scanning Calorimetry (DSC).
Provide more information of a
protein drug (HOS) in
comparison to tier 1 methods.
11. DSC measures the difference in heat flow between the sample and
the reference.
MELTING
POINT
Specific
Heat
Capacity
12. THE ADVANCED CLASS OF BIOPHYSICAL TOOLS
Tier 3 Biophysical Tools
Mass Spectroscopy (MS) and
Small Angle X-ray Scattering
(SAXS)
Tier 4 Biophysical Tools
Nuclear Magnetic Resonance
(NMR).
Spatial resolution is a term that refers to the number of pixels utilized in construction of a digital image.
The tool should be capable of providing a unique quantitative signal read out for each basic structural elements that makes up the drug that has good sensitivity with high spatial resolution resulting no or minimum overlapping of each of these unique signals. These characteristics can be broken down into four essential parts.
In developing protein biopharmaceuticals and in studying proteins in general, the most
important concept is “structure”.
HOS means protein’s three-dimensional (3D) structure.
First
is the ability to measure signals from as small a unit of the protein as possible (e.g., atomic resolution would be
ideal, but amino acid level might be acceptable, see Figure 3.2).
Second is the ability to detect all
such signals emitted from a single molecule; essentially, we want to detect and interrogate as
much of the protein’s structure (hopefully the entire structure) as possible. Third is the ability
to separate or resolve these signals spatially in a manner using some parameter space (e.g., relative
atomic distances, wavelength, temperature, time, etc.) with minimal signal overlap. The
fourth and final characteristic would be the ability to quantitatively record all of these signals
with the highest precision (and accuracy) possible.
Tier- a series of rows.
Circular Dichroism, an absorption spectroscopy, uses circularly polarized light to investigate structural aspects of optically active chiral media. It is mostly used to study biological molecules, their structure, and interactions with metals and other molecules.
Analytical ultracentrifugation is an analytical technique which combines an ultracentrifuge with optical monitoring systems.
In the last few years, it has been recognized that the standard biophysical tools (i.e., tier 1 and 2 tools) have significant limitations in their ability to detect small changes in a biopharmaceutical’s HOS (i.e., secondary and tertiary structure in the monomeric form of these drugs) due to the global nature of the information typically provided.
As a result, the task of finding a small difference between different samples of the same protein drug requires the ability to detect a difference between two very large signals. Unless these signals are acquired with high precision and accuracy, the ability to detect small differences will be significantly compromised.
Consequently, only major changes in a biopharmaceutical’s HOS can be detected with these low-resolution biophysical tools. Unfortunately, potential biophysical techniques that might help improve this situation involve high expertise, require more expensive instrumentation, are more complex to operate and require long-term data analysis commitment. Such attributes have not made these advanced biophysical tools conducive (at this time) for use in the process development area of the biopharmaceutical industry. However, this situation is changing, and a significant amount of interest and effort is mounting to explore and improve this category of biophysical methods. Thus, methods are beginning to appear (some of which are described in tier 3 and 4) that are now more user friendly, and capable of making significant contributions to improve our ability to do a better job in characterizing biopharmaceuticals.
In general, tier 1 tools correspond to those with low resolution, whereas tier 4 tools correspond to those capable of achieving high resolution.
The biophysical tools in tier 1 and 2 have then been placed into a general grouping that we believe finds widest use throughout the biopharmaceutical industry as the “standard” biophysical tools. The biophysical tools in tier 3 and 4 have been placed into an advanced grouping, which represents the tools the industry is beginning to look at more seriously at for providing a more detailed picture of the protein drugs. Nuclear magnetic resonance (NMR), hydrogen deuterium exchange with mass spectroscopy detection MS (H/DX), small angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), analytical ultracentrifugation (AUC), higher order structure (HOS), circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), size-exclusion chromatography (SEC), static light scattering/dynamic light scattering (SLS/DLS).
