Sedimentation velocity, light scattering, and dynamic light scattering are three methods for analyzing protein aggregates of various sizes and types. Sedimentation velocity provides high resolution of aggregate sizes but has low throughput. Light scattering coupled with size exclusion chromatography provides absolute molecular weights but relies on SEC limitations. Dynamic light scattering is highly sensitive to large aggregates and precursors of visible particulates. Each method has strengths and weaknesses, and no single method can detect all aggregate species present in a sample.
Measuring Comparability of Conformation, Heterogeneity and Aggregation with C...KBI Biopharma
"Measuring Comparability of Conformation, Heterogeneity, and Aggregation with Circular Dichroism and Analytical Ultracentrifugation", invited talk, State of the Art Methods for the Characterization of Biological Products and Assessment of Comparability, NIH, June 2003
Measuring Comparability of Conformation, Heterogeneity and Aggregation with C...KBI Biopharma
"Measuring Comparability of Conformation, Heterogeneity, and Aggregation with Circular Dichroism and Analytical Ultracentrifugation", invited talk, State of the Art Methods for the Characterization of Biological Products and Assessment of Comparability, NIH, June 2003
Benefits of NexION 300D ICP-MS reaction mode in removing the Gd interference ...PerkinElmer, Inc.
This work describes a case study involving the analysis of selenium in blood serum, where the ability to remove the gadolinium (Gd) doubly-charged interference is demonstrated. 011184_01
Monitoring of the Sulfur and Nano Silver in Water by Method of Laser Spectros...IJERA Editor
We developed new automatic method that combines the method of forced lumines-cence and stimulated Brillouin scattering (SBS). We used the method for the pathogens monitoring in water supply system earlier. We have researched spectral peaks of gases and show that method allows determining H2S content with 0.01% accuracy and determining the content constituents of nano silver in water.
Presentation at the ESPP workshop Pharmaceuticals in sewage biosolids, Malmo, 27-10-2016
European Sustainable Phosphorus Platform (ESPP)
www.phosphorusplatform.eu
Sustainable management of nutrients is crucial for agriculture, food, industry, water and the environment. ESPP brings together companies and stakeholders to address the Phosphorus Challenge and its opportunities for the circular economy.
Countries:
Austria AT
Belgium BE
Bulgaria BG
Cyprus CY
Czech Republic CZ
Germany DE
Denmark DK
Estonia EE
Spain ES
Finland FI
France FR
Greece EL
Hungary HU
Ireland IE
Italy IT
Lithuania LT
Luxembourg LU
Latvia LV
Malta MT
Netherlands NL
Poland PL
Portugal PT
Romania RO
Sweden SE
Slovenia SI
Slovakia SK
United Kingdom UK
Switzerland CH
Phosphorus:
Fosfor
Fosfor
Fòsfòr
Фосфор
Fosfor
Фосфор
Fosfor
Fosfor
Фосфор
Фосфор
Fosforas
Fosfors
Fuosfuors
Fosfor
Ffуsfforws
Fosfar
Fosfaras
Fosfaar
Fosforus
Φωσφορος
Ֆոսֆոր
Fosfor
Fosfor
Фосфор
Фосфор
ফসফরাস
فسفر
ફૉસ્ફરસનો
फास्फोरस
Fosfor
Fosfori
Foszfor
Фосфор
Фосфор
Паликандур
Fosfor
Fosfor
Фосфор
Фосфор
Фосфор
Фосфор
Fosfor
فوسفور
Fosfor
Fosforoa
ფოსფორი
[fūsfūr]
זרחן
Fosfru
Lìn
リン
인
ฟอสฟอรัส
Photpho
磷
Posporo
Fosfor
Pūtūtae-whetū
Fosforus
ഫോസ്ഫറസ്
பொஸ்பரசு
Fosofo
Fosforase
Posfori
Fósforo
Phusphuru
Fosforimi
Fosforo
Fosforon
Pesticium
Benefits of NexION 300D ICP-MS reaction mode in removing the Gd interference ...PerkinElmer, Inc.
This work describes a case study involving the analysis of selenium in blood serum, where the ability to remove the gadolinium (Gd) doubly-charged interference is demonstrated. 011184_01
Monitoring of the Sulfur and Nano Silver in Water by Method of Laser Spectros...IJERA Editor
We developed new automatic method that combines the method of forced lumines-cence and stimulated Brillouin scattering (SBS). We used the method for the pathogens monitoring in water supply system earlier. We have researched spectral peaks of gases and show that method allows determining H2S content with 0.01% accuracy and determining the content constituents of nano silver in water.
Presentation at the ESPP workshop Pharmaceuticals in sewage biosolids, Malmo, 27-10-2016
European Sustainable Phosphorus Platform (ESPP)
www.phosphorusplatform.eu
Sustainable management of nutrients is crucial for agriculture, food, industry, water and the environment. ESPP brings together companies and stakeholders to address the Phosphorus Challenge and its opportunities for the circular economy.
Countries:
Austria AT
Belgium BE
Bulgaria BG
Cyprus CY
Czech Republic CZ
Germany DE
Denmark DK
Estonia EE
Spain ES
Finland FI
France FR
Greece EL
Hungary HU
Ireland IE
Italy IT
Lithuania LT
Luxembourg LU
Latvia LV
Malta MT
Netherlands NL
Poland PL
Portugal PT
Romania RO
Sweden SE
Slovenia SI
Slovakia SK
United Kingdom UK
Switzerland CH
Phosphorus:
Fosfor
Fosfor
Fòsfòr
Фосфор
Fosfor
Фосфор
Fosfor
Fosfor
Фосфор
Фосфор
Fosforas
Fosfors
Fuosfuors
Fosfor
Ffуsfforws
Fosfar
Fosfaras
Fosfaar
Fosforus
Φωσφορος
Ֆոսֆոր
Fosfor
Fosfor
Фосфор
Фосфор
ফসফরাস
فسفر
ફૉસ્ફરસનો
फास्फोरस
Fosfor
Fosfori
Foszfor
Фосфор
Фосфор
Паликандур
Fosfor
Fosfor
Фосфор
Фосфор
Фосфор
Фосфор
Fosfor
فوسفور
Fosfor
Fosforoa
ფოსფორი
[fūsfūr]
זרחן
Fosfru
Lìn
リン
인
ฟอสฟอรัส
Photpho
磷
Posporo
Fosfor
Pūtūtae-whetū
Fosforus
ഫോസ്ഫറസ്
பொஸ்பரசு
Fosofo
Fosforase
Posfori
Fósforo
Phusphuru
Fosforimi
Fosforo
Fosforon
Pesticium
• It is the combination of liquid chromatography and the mass spectrometry.
• Liquid chromatography-mass spectrometry (LC-MS) is an analytical chemistry
technique that combines the physical separation capabilities of liquid
chromatography with the mass analysis capabilities of mass spectrometry.
• The combination of these two powerful techniques gives the chemical analyst the
ability to analyze virtually any molecular species; including, thermally labile, non
volatile, and high molecular weight species.
Analytical Ultracentrifugation of protein.DiNa Amin
Ultracentrifuge is a high-speed centrifuge for separating microscopic and sub-microscopic materials to determine the sizes and molecular weights of colloidal and other small particles.
Is Any Measurement Method Optimal for All Aggregate Sizes and Types? KBI Biopharma
The AAPS Journal 2006; 8 (3) Article 65 (http://www.aapsj.org).
Themed Issue: Proceedings of the 2005 AAPS Biotec Open Forum on Aggregation of Protein Therapeutics
Guest Editor - Steve Shire
Is Any Measurement Method Optimal for All Aggregate Sizes and Types?
Submitted: January 24 , 2006 ; Accepted: June 22 , 2006 ; Published: September 8 , 2006
John S. Philo 1
1 Alliance Protein Laboratories, Thousand Oaks, CA
Dynamic light scattering (DLS) is a technique in physics that can be used to determine the size distribution profile of small particles in suspension or polymers in solution.
Other names are
Photon correlation spectroscopy
Quasi-elastic light scattering.
Integration of Cell Line and Process Development to Expedite Delivery of Bisp...KBI Biopharma
Authored and Presented by: Dane A. Grismer, Yogender K. Gowtham, Srivatsan Gopalakrishnan, David. W. Chang,
Niket Bubna, Ph.D., and Sigma S. Mostafa, Ph.D.
Host Cell Protein Analysis by Mass Spectrometry | KBI BiopharmaKBI Biopharma
Host Cell Protein Analysis by Mass Spectrometry. Originally presented at the 2018 Sciex Users Meeting by Michael J Nold, Ph.D., Mass Spectrometry Core Facility at KBI Biopharma.
Handling High Titer Processes and Strategies for DSP Facility Fit | KBI Biop...KBI Biopharma
Handling High Titer Processes and Strategies for DSP Facility Fit. Originally presented at BioProcess International 2018 by Christopher Miller, Senior Scientist, Downstream Process Development, KBI Biopharma.
Octet Potency Assay: Development, Qualification and Validation StrategiesKBI Biopharma
Octet Potency Assay: Development, Qualification and
Validation Strategies
Carson Cameron, Brendan Peacor, Nathan Oien, Andrew Cheeseman, and Jimmy Smedley, KBI Biopharma, Durham, NC
John Laughlin, and David O. Apiyo, ForteBio, Fremont, CA
HIV Vaccines Process Development & Manufacturing - Pitfalls & PossibilitiesKBI Biopharma
Originally presented at the HIV Vaccine Manufacturing Workshop –July 19th& 20th, 2017 by Abhinav A. Shukla, Ph.D.Senior Vice PresidentDevelopment & ManufacturingKBI Biopharma, Durham NC
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
Adv. biopharm. APPLICATION OF PHARMACOKINETICS : TARGETED DRUG DELIVERY SYSTEMSAkankshaAshtankar
MIP 201T & MPH 202T
ADVANCED BIOPHARMACEUTICS & PHARMACOKINETICS : UNIT 5
APPLICATION OF PHARMACOKINETICS : TARGETED DRUG DELIVERY SYSTEMS By - AKANKSHA ASHTANKAR
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
Basavarajeeyam is an important text for ayurvedic physician belonging to andhra pradehs. It is a popular compendium in various parts of our country as well as in andhra pradesh. The content of the text was presented in sanskrit and telugu language (Bilingual). One of the most famous book in ayurvedic pharmaceutics and therapeutics. This book contains 25 chapters called as prakaranas. Many rasaoushadis were explained, pioneer of dhatu druti, nadi pareeksha, mutra pareeksha etc. Belongs to the period of 15-16 century. New diseases like upadamsha, phiranga rogas are explained.
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
Basavarajeeyam is a Sreshta Sangraha grantha (Compiled book ), written by Neelkanta kotturu Basavaraja Virachita. It contains 25 Prakaranas, First 24 Chapters related to Rogas& 25th to Rasadravyas.
2. Outline
Quickly review some basic facts about
aggregate sizes and types
Basic principles and application examples
for 3 methods
1. sedimentation velocity
2. classical light scattering used with SEC
3. batch-mode dynamic light scattering
3. The word “aggregate” covers a wide spectrum
of types and sizes of associated states
1. rapidly-reversible non-covalent small
oligomers (dimer, trimer, tetramer…)
2. irreversible non-covalent oligomers
3. covalent oligomers (e.g. disulfides)
4. “large” aggregates (> 10-mer)
could be reversible if non-covalent
5. “very large” aggregates (diameter ~50 nm
to 3 μm)
could be reversible if non-covalent
6. visible particulates
probably irreversible
4. Aggregates have a spectrum of lifetimes
rates of non-covalent association and dissociation (half-
times) can vary from milliseconds to days
metastable oligomers with dissociation rates of hours to
days occur fairly frequently
for an antibody example see J.M.R. Moore et al. (1999)
Biochemistry 38: 13960-13967
see also Philo, J.S. (2006) AAPS Journal, in press
many common analytical methods will detect only the
longer-lived species
it may take hours to days for a protein to re-equilibrate
its association after a change in concentration, solvent
conditions or temperature
5. Our analytical challenge
1. Any protein sample may contain aggregates
with a wide range of sizes, types, and lifetimes
2. Any one analysis method may not detect all
the aggregate sizes or types that are present
3. The measurement itself may perturb the
aggregate distribution that was initially present
dilution may dissociate reversible aggregates
change of solvent conditions may dissociate or increase
aggregates
adsorption or filtration effects may remove aggregates
7. The fundamentals of sedimentation velocity
6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0
Radius (cm)
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
Absorbance
6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0
Radius (cm)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Absorbance
centrifugal
force
diffusion
← meniscus
The sedimentation coefficient
is determined from the
boundary motion over time. It
depends on both molecular
weight and molecular shape.
cell base →
friction
←regionofsolute
depletion
boundary
8. High resolution analysis of a highly stressed antibody
sample resolves 6 aggregate peaks plus 2 fragments
0 2 4 6 8 10 12 14 16 18 20 22 24
0.0
0.2
0.4
0.6
0.8
1.0
heptamer,0.1%
hexamer,0.4%
pentamer1.4%
tetramer5.3%
trimer14.6%
dimer30.6%
main peak (monomer), 45.5%
?HLhalfmolecule,0.8%
?freelightchain,1.4%
c(s),normalized(totalarea=1)
sedimentation coefficient (Svedbergs)
9. The peril: c(s) distributions are also often
misunderstood
1. the effective resolution goes down as the fraction of
minor peaks goes down
2. the resolution you can achieve for a 150 kDa antibody
is much greater than for a 20 kDa cytokine
3. in general it is not possible to uniquely assign a
stoichiometry to each aggregate peak
4. the nature of the noise (variability) is very different
than in chromatography
5. for reversibly associating proteins the peaks probably
do not represent individual molecular species
10. This interferon-β sample is 13.7% non-covalent aggregate;
by the standard SEC method it would be pure monomer
0 2 4 6 8 10
0
1
2
3
4
5
6
7
0 2 4 6 8 10
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
IFN-β in 5 mM glycine, pH 3, 86.3% main peak
c(s)
sedimentation coefficient (Svedbergs)
11. 0
1
2
3
0.0
0.5
1.0
1.5
0 8 16 24 32 40
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
no salt
c(s)
+50 mM NaCl
c(s)
+150 mM NaCl
c(s)
sedimentation coefficient (Svedbergs)
0 2 4 6 8 10
0.00
0.05
0.10
0.15
20X expanded
0 2 4 6 8 10
0.00
0.05
0.10
0.15
20X expanded
Adding NaCl to interferon-β formulations leads to a broad
distribution of non-covalent aggregates out to ~100-mers
12. Strengths of sedimentation velocity
1. high resolution (often better than SEC)
2. covers very large range of masses in a single
experiment (much larger than SEC)
3. detects both covalent and non-covalent aggregates
4. generally can be done directly in formulation buffers
Tween and high levels of sugars do cause some
interference
5. little dilution of sample (~25%)
6. absolute method; requires no molecular standards
7. strong theoretical background; “first principles”
method
13. Weaknesses of sedimentation velocity
1. low throughput (3-7 samples/day)
2. equipment and data analysis not automated like
HPLC; labor intensive
3. expensive equipment (~250-300 K$)
4. requires substantial training
5. never been validated for lot release
Sedimentation velocity can not replace SEC, but it
is an excellent tool to test whether SEC is missing
important features. It can also serve as a “gold
standard” to help improve SEC methods.
15. Typical setup for size-exclusion chromatography
with on-line light scattering detection
light scattering
detector
absorbance
detector
refractive index
detector
size-exclusion
column
injectorpumpsolvent
16. Getting molecular mass from static light
scattering: the basic idea
the light scattering signal is proportional to the
product c × M
we measure c simultaneously with a UV or RI
detector
then the ratio of the scattering to concentration
signals will be proportional to M
masses obtained this way are absolute, and
independent of conformation and elution position
17. Demonstrating that scattering is independent of elution position
and molecular conformation: the ratio of LS to RI signals is the
same whether the protein is folded or unfolded
18 20 22 24 26 28
signal(arbitraryunits)
LS
RI
LS
RI
Native
RNase
Unfolded (reduced &
carboxymethlyated)
RNase
Retention Time (min)
18. An example for an Fc-fusion protein: the aggregate signals are
much stronger in 90° scattering than in the UV chromatogram
scattering intensity RI
elution volume (ml)
5.0 6.0 7.0 8.0 9.0 10.0 11.0
relativescale
0.0
0.2
0.4
0.6
0.8
1.0
19. “Oligomer hunting”: display the absolute molecular
weight from LS in units of monomers
6 7 8 9 10 11
0
1
2
3
4
5
6
7
8
9
10
massratiorelativetomainpeak
elution volum e (m l)
20. This antibody sample has traces of dimer and trimer
12 13 14 15 16 17 18
Elution Volume (ml)
0.0
1.0
2.0
3.0
4.0
5.0
relativemassfromLS/UV
relative mass (LS/UV) UV (arb units)
21. A different lot contains more higher oligomers, and they
are so sticky that even dimer is no longer resolved
11 12 13 14 15 16 17 18
Elution Volume (ml)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
relativemassfromLS/UV
relative mass (LS/UV) UV (arb units)
22. volume (ml)
5.0 6.0 7.0 8.0 9.0 10.0
relativescale
0.0
0.2
0.4
0.6
0.8
1.0
This highly stressed sample of a VaxGen test antigen
showed high levels of an SEC peak eluting near the
position expected for a dimer
monomer
dimer?
large
aggregates
23. However SEC-MALLS immediately shows that alleged
aggregate is actually an altered form of monomer!
molar mass vs. volume
volume
5.0 6.0 7.0 8.0 9.0 10.0
molarmass(g/mol)
5
1.0x10
6
1.0x10
7
1.0x10
24. Strengths of SEC + classical LS
1. absolute molecular mass, independent of
conformation or elution position
2. gives us at least an average mass for the
“aggregate” fraction near the exclusion limit
3. helps tell us whether our chromatography is really
working properly
4. high throughput, low cost (comparable to the HPLC
it is used with), fairly easy
5. absolute method; requires no molecular standards
6. strong theoretical background; “first principles”
method
25. Weaknesses of SEC + classical LS
1. it inherits all the problems of SEC (change in
aggregate distribution from dilution, change in buffer,
adsorption/filtration, etc.)
2. while it is very sensitive to high MW aggregates,
quantitation of % by weight still relies on the
concentration detector (RI or UV)
3. particles shed from columns may obscure the elution
region near the column’s exclusion limit
4. good signal/noise may require larger injection amounts
than are normally used in standard SEC
26. Batch-mode dynamic light scattering
(DLS)
also known as quasi-elastic light
scattering (QELS) or photon
correlation spectroscopy (PCS)
27. One particularly vexing type of aggregation is “snow”
(a.k.a. “white amorphous material” [WAM] or “floaters”)
• may only appear after many months
• often a nucleation-controlled reaction
• often ≤ 0.01% of total protein
28. ⇒
When this happens our valuable therapeutic
protein can only be used for…
Dynamic scattering is one of the few tools that may be
able to detect the precursors that eventually form ‘snow’
29. Dynamic light scattering: the basic idea
1. In dynamic scattering we measure the
fluctuations in scattering intensity (~100 ns to
30 ms)
2. The time scale of those fluctuations depends
on the diffusion coefficient of the
macromolecule, which in turn depends on its
size
3. As in classical LS, the scattering intensity is
proportional to M, so the sensitivity to very
large aggregates is very high
30. Typically the data are transformed into a distribution of
hydrodynamic radius; this distribution shows 3 peaks
2.16 nm, 79.0% of intensity
92.3 nm,
13.3% of intensity
6.58 nm,
7.8% intensity
0.015% wt.
99.1% by weight
0.9% wt.
31. Two key weaknesses of DLS
1. Low resolution
two species are not resolved as separate peaks
unless their radii differ ~2-fold (~8-fold in mass)
consequently DLS is generally not useful to detect
or quantify small oligomers (dimer-octamer)
2. Poor quantitation of weight fractions
Usually at best the reproducibility of weight
fractions is only +/- a factor of 2
There is no universally-accepted standard
algorithm to calculate weight fractions; different
methods can give quite divergent results
32. Here is an example for a small peptide that
forms visible thread-like particles
7.9% intensity,
99.998% by weight
92.1% intensity,
0.002% by weight
species in the ~20-400
nm size range are often
precursors or nuclei for
formation of visible
particulates
33. In our hands DLS has been the most effective
tool for detecting precursors of visible particulates
1. Useful for qualitative assessment of different
formulations, ‘good’ vs. ‘bad’ lots
2. Useful to track where in the manufacturing process
damage to the protein is occurring
in one case tracked to specific pump
in another case to viral filtration step
3. Useful to detect contaminant particles that can serve
as nuclei onto which protein aggregates
(heterogeneous nucleation)
silicones
glass particles from vials
vacuum pump oil from lyophilizers
34. Strengths of DLS
1. high sensitivity to large aggregates that may be
immunogenic and/or precursors to visible
particulates
2. covers an enormous range of sizes in one analysis
(range of mass > 109)
3. done at equilibrium; theoretically senses all forms of
aggregates
4. batch mode
no dilution
no change of solvent conditions
no loss of species to frit or column matrix