This document discusses challenges in analyzing protein aggregates using analytical methods. It provides examples of unusual aggregation phenomena observed:
1) An "aggregate" peak seen by SEC in a stressed sample was shown to be a partially denatured monomer using SEC-MALLS and sedimentation velocity.
2) Freeze/thaw stress generated transient, metastable oligomers in a protein that were difficult to detect by SEC due to their stickiness. Sedimentation velocity detected a much higher level of aggregates.
3) Different analytical methods can perturb aggregate distributions in different ways by dissociating or creating new aggregates. Method selection depends on the aggregate properties and no single method is optimal in all cases.
CHAS 31: Encoding reactive chemical hazards and incompatibilities in an alert...NextMove Software
Of the many chemical reactions performed by synthetic chemists in the pharmaceutical industry and academia, some are potentially more hazardous than others. Fortunately, best practices, compliance and education helps ensure that incidents are rare, but as highlighted by the recent explosion and building evacuation at two UK universities in March 2015, constant vigilance is necessary to ensure a safe work environment. The primary problem is not that chemical safety information, for example from MSDS/SDS data sheets, Bretherick's Handbook or the internet, is readily available, but that the volume of such information makes it difficult for an experimentalist to identify relevant risks in a timely manner.
In this talk, we describe our attempts to encode the Environmental Protection Agency's (EPA's) guidance entitled 'A Method for Determining Compatibility of Hazardous Waste', 1980, in an XML file format. Typical current state-of-the-art methods for alerting potential chemical safety hazards, for example in ELNs, simply annotate reactants with codes extracted from their MSDS/SDS data sheets, such as Global Harmonized System of Classification and Labelling of Chemicals (GHS) or EU R-phrases/S-phrases, leaving the chemist to manually assess whether any of the described incompatibilities is relevant. In this work, we use combinations of SMARTS patterns (for chemical classes) and InChIs (for specific molecules), to capture known reagent incompatibilities, that may be safe in isolation. Specific alerts describe documented incompatibilities between compounds (e.g. acetone and H2O2) while more general alerts can capture known or inferred incompatibilities between functional groups (e.g. ketones and peroxides). The encoding is hierarchical allowing only the most relevant warnings to be triggered. Alerts are encoded in a flexible XML format, facilitating extension and exchange. Advanced features of this XML format, include the ability to specify reactant quantities, and the use of predicted properties (such as of products) in rules.
Metabolomics is often described as the study of “the complete set of low molecular weight intermediates, which are context dependent, varying according to the physiology, developmental or pathological state of the cell, tissue, organ or organism”. In fact, metabolomics is a new term for an old science in which classical biochemical concepts are investigated. New and unique to the current research that is being conducted is the combination with genomics information and full system biology. In this refocus we will discuss the challenges in today's metabolomics research and how to address them
CHAS 31: Encoding reactive chemical hazards and incompatibilities in an alert...NextMove Software
Of the many chemical reactions performed by synthetic chemists in the pharmaceutical industry and academia, some are potentially more hazardous than others. Fortunately, best practices, compliance and education helps ensure that incidents are rare, but as highlighted by the recent explosion and building evacuation at two UK universities in March 2015, constant vigilance is necessary to ensure a safe work environment. The primary problem is not that chemical safety information, for example from MSDS/SDS data sheets, Bretherick's Handbook or the internet, is readily available, but that the volume of such information makes it difficult for an experimentalist to identify relevant risks in a timely manner.
In this talk, we describe our attempts to encode the Environmental Protection Agency's (EPA's) guidance entitled 'A Method for Determining Compatibility of Hazardous Waste', 1980, in an XML file format. Typical current state-of-the-art methods for alerting potential chemical safety hazards, for example in ELNs, simply annotate reactants with codes extracted from their MSDS/SDS data sheets, such as Global Harmonized System of Classification and Labelling of Chemicals (GHS) or EU R-phrases/S-phrases, leaving the chemist to manually assess whether any of the described incompatibilities is relevant. In this work, we use combinations of SMARTS patterns (for chemical classes) and InChIs (for specific molecules), to capture known reagent incompatibilities, that may be safe in isolation. Specific alerts describe documented incompatibilities between compounds (e.g. acetone and H2O2) while more general alerts can capture known or inferred incompatibilities between functional groups (e.g. ketones and peroxides). The encoding is hierarchical allowing only the most relevant warnings to be triggered. Alerts are encoded in a flexible XML format, facilitating extension and exchange. Advanced features of this XML format, include the ability to specify reactant quantities, and the use of predicted properties (such as of products) in rules.
Metabolomics is often described as the study of “the complete set of low molecular weight intermediates, which are context dependent, varying according to the physiology, developmental or pathological state of the cell, tissue, organ or organism”. In fact, metabolomics is a new term for an old science in which classical biochemical concepts are investigated. New and unique to the current research that is being conducted is the combination with genomics information and full system biology. In this refocus we will discuss the challenges in today's metabolomics research and how to address them
ADME – A Key To An Effective And Safe Drug – Selvita.pdflizseyi
ADME is an acronym used in pharmacology. It stands for Absorption, Distribution, Metabolism, and Excretion. In short, these are the processes that take place in our body in the context of foreign substances, including drugs. It is how drugs are absorbed, transported around our body, metabolized, and excreted that affects whether a drug is effective (reaches its destination) and safe (does not cause side effects).
Physicochemical Profiling In Drug ResearchBrian Bissett
Physicochemical and Biological Profiling in Drug Research ElogD(7.4) 20,000 Compounds Later: Refinements, Observations and Applications
Franco Lombardo, Marina Y. Shalaeva, Brian D. Bissett and Natalya Chistokhodova.
Molecular Properties Group, PGRD Groton Laboratories, Groton, CT 06340, U.S.A.
Comparative Essay - 10 Examples, Format, Pdf Examples. Writing A Comparative Essay Step 1 - Choose Your Subject. Sample comparison essay. 100 Great Compare and Contrast Essay Topics .... 005 Essay Example An Of Compare And Contrast Comparison Ideas Thatsnotus. How to write an introduction to a comparison essay Oneonta .... 002 Compare And Contrast Essay Sample Paper Comparecontrast Thesis .... Eng 2 Comparison Essay. How to write a good comparison essay. custom writing online. 019 Comparison Essay Sample Thatsnotus. What is comparative essay. How To Write An Effective Introduction For .... 013 Comparison Essay Outline Format Template 474624 Thatsnotus. Comparison essay. Comparison Essay.docx Essays Thesis. Scholarship essay: Comparative analysis essay example. Comparing and Contrasting - The Writing Center - How to Write a Compare .... Writing A Comparative Essay : Sample Comparative Essay Format. Comparison essays examples. 2 Comparison Essay Examples That Make .... Writing A Comparative Essay How to write a perfect comparative essay .... Writing an comparison essay. How to Write a Comparative Essay: Step-by-Step Structure - Ca.EduBirdie.com. 9 Comparative Essay Samples - Free PDF Format Download Examples. How to Write a Compare and Contrast Essay Outline Point-By-Point With .... Comparison Essay Assignment. 021 Comparison Essays Outline Format 2 Thatsnotus. 006 Essay Example Comp
ElogPoct: A Tool for Lipophilicity Determination in Drug DiscoveryBrian Bissett
ElogPoct: A Tool for Lipophilicity Determination in Drug Discovery
Franco Lombardo,Marina Y. Shalaeva, Karl A. Tupper,Feng Gao, and Michael H. Abraham
Molecular Properties Group and Mathematical and Statistical Sciences Group, Central Research Division,
Pfizer Inc., Groton, Connecticut 06340, and Department of Chemistry, University College London, 20 Gordon Street,
London, United Kingdom WC1H OAJ
A seminar report on the chemical frontiers of living matter seminar series - ...Glen Carter
This seminar report highlights a select few presentations of cutting-edge research being done in various labs across the Paris Science et Lettre (PSL) network.
Lab report that discusses the antigen-antibody precipitation reaction using the Ouchterlony Double Diffusion Technique.
Created by: Annisa Hayatunnufus
Bachelor of Pharmacy
Management & Science University
Investigating cellular metabolism with the 3D Cell ExplorerMathieuFRECHIN
Long-term imaging of fine dynamics of cellular organelles is today’s biggest challenge in cell biology
(Frechin et al., 2015; Kruse & Jülicher, 2005; Kueh, Champhekhar, Nutt, Elowitz, & Rothenberg, 2013;
Skylaki, Hilsenbeck, & Schroeder, 2016). The goal is to acquire not only snapshots of dynamic biological
systems, but to actually see processes unfolding over time in term of spatial and morphological
changes and biological outcome (Muzzey, Gómez-Uribe, Mettetal, & van Oudenaarden, 2009).
Imaging over time is of utmost importance in the study of key organelles implicated in cellular
metabolism: mitochondria and lipid droplets. The current method of choice in high-content live
imaging approaches is fluorescence microscopy. However, fluorescence microscopy induces
phototoxicity when the sample is stimulated at various wavelengths. This stress induces cellular
damages via radical-induced cellular structure alterations, which limits live imaging possibilities.
Therefore, with the current live cell imaging strategies a tradeoff must be found between short
live cell imaging with high-frequency acquisition or long-term live cell imaging with low-frequency
acquisition.
On one hand, high-frequency acquisition induces a lot of phototoxic stress and, if successful, a
researcher might observe fine dynamics but cannot be sure that they have not been perturbed
by the imaging process. On the other hand, low-frequency acquisition might be more sustainable,
however, fine dynamics are lost, while the observed phenomenon, to a lesser extent, could likewise
be perturbed by the imaging process.
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.
ADME – A Key To An Effective And Safe Drug – Selvita.pdflizseyi
ADME is an acronym used in pharmacology. It stands for Absorption, Distribution, Metabolism, and Excretion. In short, these are the processes that take place in our body in the context of foreign substances, including drugs. It is how drugs are absorbed, transported around our body, metabolized, and excreted that affects whether a drug is effective (reaches its destination) and safe (does not cause side effects).
Physicochemical Profiling In Drug ResearchBrian Bissett
Physicochemical and Biological Profiling in Drug Research ElogD(7.4) 20,000 Compounds Later: Refinements, Observations and Applications
Franco Lombardo, Marina Y. Shalaeva, Brian D. Bissett and Natalya Chistokhodova.
Molecular Properties Group, PGRD Groton Laboratories, Groton, CT 06340, U.S.A.
Comparative Essay - 10 Examples, Format, Pdf Examples. Writing A Comparative Essay Step 1 - Choose Your Subject. Sample comparison essay. 100 Great Compare and Contrast Essay Topics .... 005 Essay Example An Of Compare And Contrast Comparison Ideas Thatsnotus. How to write an introduction to a comparison essay Oneonta .... 002 Compare And Contrast Essay Sample Paper Comparecontrast Thesis .... Eng 2 Comparison Essay. How to write a good comparison essay. custom writing online. 019 Comparison Essay Sample Thatsnotus. What is comparative essay. How To Write An Effective Introduction For .... 013 Comparison Essay Outline Format Template 474624 Thatsnotus. Comparison essay. Comparison Essay.docx Essays Thesis. Scholarship essay: Comparative analysis essay example. Comparing and Contrasting - The Writing Center - How to Write a Compare .... Writing A Comparative Essay : Sample Comparative Essay Format. Comparison essays examples. 2 Comparison Essay Examples That Make .... Writing A Comparative Essay How to write a perfect comparative essay .... Writing an comparison essay. How to Write a Comparative Essay: Step-by-Step Structure - Ca.EduBirdie.com. 9 Comparative Essay Samples - Free PDF Format Download Examples. How to Write a Compare and Contrast Essay Outline Point-By-Point With .... Comparison Essay Assignment. 021 Comparison Essays Outline Format 2 Thatsnotus. 006 Essay Example Comp
ElogPoct: A Tool for Lipophilicity Determination in Drug DiscoveryBrian Bissett
ElogPoct: A Tool for Lipophilicity Determination in Drug Discovery
Franco Lombardo,Marina Y. Shalaeva, Karl A. Tupper,Feng Gao, and Michael H. Abraham
Molecular Properties Group and Mathematical and Statistical Sciences Group, Central Research Division,
Pfizer Inc., Groton, Connecticut 06340, and Department of Chemistry, University College London, 20 Gordon Street,
London, United Kingdom WC1H OAJ
A seminar report on the chemical frontiers of living matter seminar series - ...Glen Carter
This seminar report highlights a select few presentations of cutting-edge research being done in various labs across the Paris Science et Lettre (PSL) network.
Lab report that discusses the antigen-antibody precipitation reaction using the Ouchterlony Double Diffusion Technique.
Created by: Annisa Hayatunnufus
Bachelor of Pharmacy
Management & Science University
Investigating cellular metabolism with the 3D Cell ExplorerMathieuFRECHIN
Long-term imaging of fine dynamics of cellular organelles is today’s biggest challenge in cell biology
(Frechin et al., 2015; Kruse & Jülicher, 2005; Kueh, Champhekhar, Nutt, Elowitz, & Rothenberg, 2013;
Skylaki, Hilsenbeck, & Schroeder, 2016). The goal is to acquire not only snapshots of dynamic biological
systems, but to actually see processes unfolding over time in term of spatial and morphological
changes and biological outcome (Muzzey, Gómez-Uribe, Mettetal, & van Oudenaarden, 2009).
Imaging over time is of utmost importance in the study of key organelles implicated in cellular
metabolism: mitochondria and lipid droplets. The current method of choice in high-content live
imaging approaches is fluorescence microscopy. However, fluorescence microscopy induces
phototoxicity when the sample is stimulated at various wavelengths. This stress induces cellular
damages via radical-induced cellular structure alterations, which limits live imaging possibilities.
Therefore, with the current live cell imaging strategies a tradeoff must be found between short
live cell imaging with high-frequency acquisition or long-term live cell imaging with low-frequency
acquisition.
On one hand, high-frequency acquisition induces a lot of phototoxic stress and, if successful, a
researcher might observe fine dynamics but cannot be sure that they have not been perturbed
by the imaging process. On the other hand, low-frequency acquisition might be more sustainable,
however, fine dynamics are lost, while the observed phenomenon, to a lesser extent, could likewise
be perturbed by the imaging process.
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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
Title: Sense of Smell
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 primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
Is Any Measurement Method Optimal for All Aggregate Sizes and Types?
1. E564
The AAPS Journal 2006; 8 (3) Article 65 (http://www.aapsj.org).
ABSTRACT
Protein-based pharmaceuticals exhibit a wide range of
aggregation phenomena, making it virtually impossible to
find any one analytical method that works well in all cases.
Aggregate sizes cover a range from small oligomers to
visible “snow” and precipitates, and generally only the
smaller species are reversible. It is less widely recognized
that aggregates also exhibit a broad spectrum of lifetimes,
and the lifetime has important consequences for detection
methods. The fact that the measurement itself may destroy
or create aggregates poses a major analytical challenge
and is a key determinant for method selection. Several ex-
amples of some interesting aggregation phenomena and
the analytical approaches we have used are presented. In
one case, an “aggregate” seen by SEC in stressed samples
was shown to actually be a partially denatured monomer
using both size-exclusion chromatography with online
multiangle laser light scattering (SEC-MALLS) and sedi-
mentation velocity. In a second case, freeze/thaw stress
generatestransient,metastableoligomersthatareextremely
sticky and difficult to measure by SEC. By using sedimen-
tation velocity as the “gold standard” a much improved
SEC method was developed and used to investigate the
temperature-dependent dissociation of these oligomers.
For problems with visible particulates, dynamic light scat-
tering has been effective, in our hands, at detecting the
precursors to the large, visible particles and tracking the
source of stress or damage to particular manufacturing
steps.
KEYWORDS: aggregate, oligomer, analytical ultracentrifu-
gation, sedimentation velocity, light scattering
INTRODUCTION
The measurement and control of aggregation continues
to be a major concern for biopharmaceutical products.
At Alliance Protein Laboratories it has been a privilege and
a challenge to work on aggregation issues in peptides, pro-
teins, and other macromolecules for more than 80 compa-
nies. Many clients come to us expecting that there should
be one analytical method or approach that will give a com-
plete answer and that will work in all situations and for all
of their different products. I wish it was that easy!
In working with clients I have learned that many pharma-
ceutical scientists fail to appreciate the full range of aggre-
gation phenomena that can and do occur and how that
impacts the measurement approach and the proper interpre-
tation of the data. Therefore, the first part of this article will
highlight some common misunderstandings about aggre-
gates and some of the analytical challenges we face. Then
a few specific examples of some unusual or challenging
aggregation problems will be presented, which we have
approached using light-scattering and/or analytical ultra-
centrifugation techniques.
THE WIDE SPECTRUM OF AGGREGATE SIZES
AND TYPES
There is unfortunately no uniform terminology for aggre-
gate sizes or types, but some of the different classes we
should consider are (1) rapidly reversible noncovalent small
oligomers (dimer, trimer, tetramer, and so forth); (2) irre-
versible noncovalent oligomers; (3) covalent oligomers (eg,
disulfide-linked); (4) “large” aggregates (≥ 10-mer), which
could be reversible if noncovalent; (5) “very large” aggre-
gates (diameter ~50 nm to 3 mm), which could be reversible
if noncovalent; and (6) visible particulates (“snow” or
“floaters”), which are probably irreversible.
It is of course often the case that over time the aggregates
in any particular sample evolve, typically becoming less
reversible and larger. It is also important to remember that
samples are likely to contain more than one of these types or
classes.
Many pharmaceutical scientists like to divide aggregates
into “soluble” and “insoluble” categories. However if you
ask carefully you find that these words mean very different
things to different people—to some scientists any aggregate
big enough to elute in the void volume of an SEC column is
“insoluble,” to others “insoluble” means it forms a visible
precipitate. Consequently this terminology can be far more
misleading than helpful, and I would discourage the use of
these terms.
Corresponding Author: John S. Philo, Alliance Protein
Laboratories, 3957 Corte Cancion, Thousand Oaks,
CA 91360. Tel: 805-388-1074; Fax: 805-388-7252;
E-mail: jphilo@ap-lab.com
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. Philo1
1Alliance Protein Laboratories, Thousand Oaks, CA
2. The AAPS Journal 2006; 8 (3) Article 65 (http://www.aapsj.org).
E565
Reversibility
With regard to reversibility, most scientists tend to think of
“reversible” and “irreversible” as a permanent, black versus
white distinction. In reality though there is a continuum of
states between reversible and irreversible; an aggregate
that is irreversible in one context can become reversible in
another. Factors that can affect reversibility are (1) solvent
components, including salts, sugars, and other excipients,
as well as organic modifiers (alcohols, acetonitrile); (2) pH;
(3) temperature; and (4) how long you wait.
The Time Dimension
Perhaps the most overlooked property of reversible aggre-
gates is that they have a wide spectrum of lifetimes. The rates
of association and dissociation reactions vary enormously, so
the lifetime of any one oligomer molecule may range from
milliseconds to several days. Many analytical methods,
especially those involving separation, will only detect the
longer-lived species. Further, separation methods operate on
different characteristic timescales; for example, separations
bySECtypicallyoccurinapproximately15minutes,whereas
sedimentation velocity (SV) experiments generally run for at
least several hours. Thus, aggregate lifetimes can play a very
major role in whether an aggregate will be resolved and
detected by any particular analytical method.
When we apply a separation method to a protein exhibiting
rapidly reversible self-association, there is a constant battle
between separation and reequilibration because of the law
of mass action. Under such circumstances the results often
depend on the rates of the association-dissociation reactions
as well as the equilibrium constants, and things can get
surprisingly complex. Although we may actually resolve
multiple peaks by SEC, SV, or flow field-flow fractionation
(FFFF), generally each of those peaks does not represent a
pure, individual oligomer, but rather a dynamic mixture of
multiple oligomers. When the association-dissociation rates
are slow (comparable to the rate of physical separation), in
theory it is possible to resolve more peaks than there are
species present1,2!
When the association-dissociation reactions are very slow
compared with the time scale of the separation then our
interacting system behaves like a true mixture and we can
resolve individual oligomers. Surprisingly, such reversible
but extremely slow association reactions, producing aggre-
gates we call “metastable oligomers,” seem to be fairly
common. A good example in the literature for a monoclonal
antibody was provided by Moore et al,3 but I have seen sim-
ilar phenomena in several other antibodies as well as smaller
proteins.
The good news about the existence of such metastable
oligomers is that their long lifetimes make them easy to
detect. The bad news is that when a protein exhibits
extremely slow association-dissociation that means every
sample has a long “memory” of its prior history (concentra-
tion, temperature, solvent conditions) and may require hours
or even days to reequilibrate to new conditions. This can
easily lead to confusing differences from one measurement
to another, as occurred in an Example 2.
OUR MEASUREMENTS ARE INHERENTLY
PERTURBING
One of the most critical problems in analyzing aggregates is
the simple fact that most measurement techniques at least
potentially perturb the distribution of species we are trying
to measure. Generally the problem is that the measurement
destroys or loses some of the aggregates, but new aggre-
gates can also be created by or during the measurement.
Often this is the reason regulatory agencies may ask appli-
cants to use multiple techniques (preferably orthogonal ones)
to measure aggregation.
Table 1 summarizes some mechanisms that lead to loss or
creation of aggregates and an indication of the relative size
of that problem for SEC, SV, and FFFF. (Note the informa-
tion regarding FFFF summarizes opinions given to me by
expert users, I am not a practitioner of that technique.) Cer-
tainly it is true that different scientists might assign different
weights to these issues, but the overall patterns should be
correct.
Both SEC and FFFF produce a high dilution of the sample
that will tend to dissociate the reversible aggregates; for
SV the total dilution is only about 20%. For SV another
difference is that the high molecular weight aggregates or
Table 1. Mechanisms for Loss or Creation of Aggregates by
Several Separation Methods*
Dissociation or loss of aggregates
can be caused by: SEC SV FFFF
dilution +++ + +++
change of solvent conditions +++ – ++
adsorption to surfaces +++ + ++
physical filtration (eg, column frit) +++ – –
physical disruption (eg, shear forces) ++ – –
Creation of new aggregates can be
caused by:
change of solvent conditions +++ – ++
surface or shear-induced denaturation ++ – +
concentration on surface – – +
*The number of pluses indicates the relative size of the problem for
that method.
SEC indicates size exclusion chromatography; SV, sedimentation
velocity; FFFF, flow field-flow fractionation.
3. The AAPS Journal 2006; 8 (3) Article 65 (http://www.aapsj.org).
E566
complexes are always sedimenting in the presence of the
smaller, slowly sedimenting species, and that fact can have
a significant impact on the distribution of species that is
detected for reversibly interacting systems.4
SEC is also rather notorious for filtration effects and poor
sample recovery, which often force the use of an elution
buffer containing high levels of salts and/or organic modifi-
ers, which in turn will modify the distribution of noncova-
lent aggregates. FFFF also can suffer from adsorption of
proteins to the cross-flow membrane, and consequently
some adjustment of elution solvent composition may be
needed, but these issues are generally lower than for SEC.
The ability to work with a wide range of solvent conditions
is an important strength of sedimentation velocity, but it too
has its limits and exhibits some interference from detergents
and high levels of sugars.
With regard to creation of new aggregates, generally the
biggest problem arises from changes in solvent composi-
tion. I have seen several cases where high ionic strength
SEC elution buffers caused formation of aggregates that
were not actually present in the formulated samples. Those
problems are exacerbated by the common practice of predi-
luting the SEC samples with the elution buffer.
EXAMPLE 1: AN “AGGREGATE” THAT ISN’T
ACTUALLY AN AGGREGATE
A recombinant antigen being tested for vaccine applications
exhibited some unusual and interesting behavior. When
subjected to stress it developed new early-eluting SEC
peaks, including a prominent peak near the elution position
expected for a dimer (Figure 1). To confirm that this species
is indeed a dimer I ran SEC with online multiangle classical
light-scattering detection (SEC-MALLS). To my surprise,
even before the chromatogram was complete the light-
scattering data made it obvious that this alleged “dimer”
peak was actually an altered conformation of monomer
(Figure 2). Why do I say this is obvious? Remember that the
true molecular mass for each peak is proportional to the
ratio of the signals from the light-scattering (LS) and refrac-
tive index (RI) detectors.5 Although the LS signal shows a
strongly sloping background as a result of trailing of very
large aggregates eluting near the void volume, it is nonethe-
less clear that the ratio of LS/RI peak heights for the puta-
tive dimer eluting at approximately 7.2 mL is about the
same as the LS/RI ratio for the monomer peak eluting at
approximately 8.3 mL. If this truly was a dimer, the LS/RI
ratio would be twice as large as that for monomer.
One can of course calculate the true molecular mass for each
peakfromthesedata,andthatmolecularmasschromatogram
is shown in Figure 3. The coelution of some sticky large
Figure 1. SEC chromatogram for a highly stressed sample of a
test antigen.
Figure 2. Overlay of the 90° light scattering (black) and RI
(red) chromatograms for the sample in Figure 1 after scaling
to match the peak heights for the monomer. Note that the sticky
high molecular weight aggregates are tailing out to at least
10 mL, raising the light-scattering baseline substantially
through that region.
Figure 3. Overlay of the molecular mass (points) and RI (line)
chromatograms calculated from the data in Figure 2.
4. The AAPS Journal 2006; 8 (3) Article 65 (http://www.aapsj.org).
E567
aggregates with the peak at 7.2 mL raises its apparent mass
to approximately 106 kDa, somewhat above the correct
monomer mass of approximately 80 kDa, but nonetheless it
is obvious that this species is indeed a monomer and not an
aggregate. The early elution of this monomer at 7.2 mL
implies this is a hydrodynamically expanded, partially dena-
tured form of monomer.
That interpretation was confirmed by SV. The sedimenta-
tion coefficient distribution (Figure 4) shows the presence
of both the normal native monomer at 4.60 Svedberg (S)
and a large amount of a slowly sedimenting species at 3.62
S (the expanded monomer). This partially denatured mono-
mer has higher hydrodynamic friction than the compact
native monomer, but the same mass, and therefore it sedi-
ments more slowly. This sample also contains a low mass
impurity at 2.43 S. It is unclear whether the minor peak at
4.04 S represents a true intermediate conformation or results
from dynamic interconversion of the 3.62-S and 4.60-S
conformations.
It is worth noting that altered conformations produce
changes in opposite directions for SV and SEC. In SEC an
expanded conformation elutes like a species of higher mass,
whereas in SV expanded conformations sediment more
slowly, like a species of lower mass. This is another way in
which these methods are orthogonal, and that difference can
be exploited to distinguish conformational differences from
mass differences.
EXAMPLE 2: METASTABLE NONCOVALENT
AGGREGATES
The next example concerns a recombinant glycoprotein,
Protein X. This protein is known to easily generate noncovalent
aggregates, but it is also notorious for sticking to surfaces
and therefore it is very difficult to measure those aggregates
by SEC (because of poor sample recovery). In the course of
trying to develop improved formulations for this protein we
encountered wide variations in aggregate levels as measured
by different labs and different methods. As you might
expect, some of these differences did indeed turn out to be a
result of analytical issues, but surprisingly we also found
there were real aggregation differences because of an inter-
esting transient aggregation phenomenon and differences in
sample histories.
One of the issues we were facing was that this protein can
be quite sensitive to freeze/thaw damage, making it difficult
to hold or ship the bulk drug substance. Figure 5 shows sed-
imentation velocity data for a sample stressed by 4 freeze/
thaw cycles, giving a total content of aggregates (species
sedimenting faster than the main peak) of 19.9%, distrib-
uted over at least 9 different aggregate species. Dilution to
different protein concentrations gave similar distributions
(not shown), indicating these are long-lived species not in
rapid equilibrium with the monomer.
These sedimentation velocity results were in sharp contrast
to the values returned by the standard SEC method for this
protein, which gave only approximately 4% aggregate. Why
are the results so different? As mentioned previously, this
protein is quite sticky and to get good recovery the standard
SEC method uses an elution buffer that is fairly denaturing.
To prove this was the source of the discrepancy, the same
sample that was stressed 4 times was diluted into the SEC
elution buffer and run by sedimentation velocity. Those
results (Figure 6) clearly showed that the SEC buffer caused
dissociation of most of the noncovalent aggregates, leaving
only aggregates that are either covalent or highly irrevers-
ible. This is a good example of the point made earlier that
Figure 5. Normalized sedimentation coefficient distribution for
Protein X drug substance stressed by 4 freeze/thaw cycles. The
inset shows the same data vertically amplified ~50-fold so the
minor peaks can be seen. The total aggregate content is 19.9%.
Figure 4. Sedimentation coefficient distribution for the stressed
test antigen. The distribution has been normalized so the area
under each peak gives the fraction of that species.
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E568
reversibility depends on context—these noncovalent aggre-
gates were effectively irreversible in the bulk drug substance
buffer (they did not dissociate when the samples were diluted
and run), but became reversible and dissociated quickly in
the SEC elution buffer.
Using the SV data as the “gold standard” for the true aggre-
gate levels, an improved SEC method was developed that
preserves the noncovalent aggregates and still gives good
recovery and reasonable resolution. Figure 7, A and B,
shows parallel measurements of a sample subjected to many
freeze/thaw cycles using this improved SEC method and
SV. The methods agree quite well on the fraction monomer
(63.4% by SEC, 63.2% by SV) and fairly well on the frac-
tion dimer (12.2% by SEC, 11.4% by SV). It is clear the
resolution of SV is much better for the larger aggregates,
and indeed it seems likely that the “dimer” peak in SEC is
partially contaminated by trimer, which may explain some
of the quantitative differences. Overall though we should
not really expect to get perfect quantitative agreement—
what we are looking for is a high correlation between the
methods.
The Freeze/Thaw-induced Dimer is Metastable
Despite the improved SEC method there was still difficulty
getting consistent aggregate levels for some samples. Finally
we realized that the aggregate levels can change over time
after the samples are thawed, and these changes are strongly
dependent on temperature. Figure 8, A and B, shows the
SEC and SV data for a freshly thawed sample of bulk drug
substance. Both methods indicate levels of dimer similar
to those for the sample subjected to many freeze/thaw cy-
cles (Figure 7) but significantly lower levels of larger
aggregates.
If this stock is allowed to stand at warm temperatures for
some time, however, there are dramatic shifts in the aggre-
gate distribution. Figure 9, A and B, shows SEC and SV
data for a sample thawed and then held at 29°C for 16 hours
before the measurements. This incubation produces a dra-
matic drop in the dimer content, and some reduction in the
larger aggregates, with a corresponding increase in mono-
mer content. That is, these freeze/thaw-induced aggregates
are metastable species that can dissociate and revert to
monomer, but they do so only very slowly.
Figure 10 shows that the rate of dissociation of dimer to
monomer is strongly dependent on temperature. Incubation
for 24 hours at 4°C produces very little dissociation to
monomer, but at 29°C most of the dimer is gone, and at
40°C dissociation is essentially complete. Thus, the overall
picture is that freeze/thaw events create a nonequilibrium
distribution of dimer and larger noncovalent aggregates,
and with time and the right conditions those aggregates can
dissociate to monomer. Because of these properties the
aggregate content of each sample depends on its detailed
Figure 6. Normalized sedimentation coefficient for the freeze/
thaw stressed stock in Figure 5 after dilution into the standard
SEC buffer, giving 4.0% total aggregate.
Figure 7. Comparison of the improved SEC method (A) with
sedimentation velocity data (B) for a Protein X sample subjected
to many freeze/thaw cycles.
6. The AAPS Journal 2006; 8 (3) Article 65 (http://www.aapsj.org).
E569
history (time, temperature, and presumably other solvent
conditions).
EXAMPLE 3: DEALING WITH VISIBLE
PARTICULATES (“SNOW”)
The formation of visible particulates by protein samples
can be a particularly vexing problem. Such particles may
not appear until months after manufacturing, making it
extremely difficult to track down the cause of the problem
and to be certain it will not recur. Although the particles
may be distinctly visible, the actual fraction of the protein
they represent is typically quite small, often 0.01% or less
of the total. The formation of these particles is often a
nucleation-controlled reaction—no particles appear until
the concentration of an intermediate-size particle (usually
subvisible) reaches a critical point, and then large particles
grow rapidly from these seeds. In some cases those critical
nuclei are product aggregates (so-called homogeneous
nucleation), but in other cases the critical nuclei are actu-
ally particulate contaminants introduced during manufac-
turing or from containers and closures (heterogeneous
nucleation).
So the key to tracking down the source of these particulate
problems and to predicting whether a change in formula-
tion or the manufacturing process will cure them (rather
than simply waiting to see if particles appear) is usually to
detect these critical nuclei. However the fact that the criti-
cal nuclei are often present at levels well below 0.1% by
weight creates a formidable analytical challenge. In my
experience the most useful tool for detecting the precursors
of the visible particles is dynamic light scattering (DLS),
and this approach has helped to solve this type of problem
in several cases.
Figure 8. Aggregates in a freshly thawed sample of bulk drug
substance measured by the improved SEC method (A) and by
sedimentation velocity (B). Figure 9. Aggregates in a sample of bulk drug substance that
was thawed and incubated at 29°C for 16 hours prior to
measurement. SEC data (A); sedimentation velocity (B).
7. The AAPS Journal 2006; 8 (3) Article 65 (http://www.aapsj.org).
E570
What is Dynamic Light Scattering?
Dynamic light scattering is also known as quasi-elastic light
scattering (QELS) and photon correlation spectroscopy
(PCS). In DLS the fluctuations in light-scattering intensity
as a function of time are measured, over time scales from
approximately 100 ns to approximately 30 ms, rather than
the time-averaged intensity that is studied in “classical” or
“static” light scattering. Those fluctuations are due to the
Brownian motion of the scattering particles. The time scale
of the scattering fluctuations is directly related to the trans-
lational diffusion coefficient of the scattering particles,
which in turn is related to their size. In DLS, as for classical
LS, the scattering intensity for any species is proportional to
the product of the weight concentration times its molecular
mass. Thus, the large aggregates produce very strong scat-
tering signals and the sensitivity for large species is very
high.
Another important advantage of DLS is that it can be done
as a batch-mode measurement. In that mode the samples are
at thermodynamic equilibrium (there is no dilution or physi-
cal separation) and there is no column matrix on which
aggregates can be lost. In batch mode when multiple species
(multiple hydrodynamic sizes) are present each species has
its own characteristic time scale for fluctuations. Thus in
principle it is possible to mathematically resolve and sepa-
rate the contributions from each species, and when this is
done one can generate a distribution of hydrodynamic sizes
(like a chromatogram). However, the resolution of this
mathematical separation is fairly poor.
To illustrate, Figure 11 shows the hydrodynamic radius dis-
tribution for a sample that contains some large aggregates.
This histogram plots the fraction of scattering intensity ver-
sus the hydrodynamic radius (by definition the hydrody-
namic radius of a molecule is the radius of a spherical parti-
cle that has the same diffusion coefficient as that molecule).
Note that the radius scale is logarithmic, and this measure-
ment covers an enormous range of sizes from approximately
0.1 to approximately 3000 nm (covering species differing in
molecular mass by a factor of approximately 3 × 1013!).
The size histogram for this sample shows 3 peaks at mean
radii of 2.16, 6.58, and 92.3 nm, representing 79.0%, 7.8%,
and 13.3% of the total scattering intensity, respectively.
Although the aggregate peaks represent a large fraction of
the scattering intensity, because the sensitivity increases
proportionally to the radius cubed those peaks correspond
to a very small fraction by weight (estimated as 0.9% for the
6.58-nm peak, and only 0.015% for the 92.3-nm peak). So
this approach can easily detect possible precursors of visible
particles even though they represent less than 0.01% by
weight!
Figure 12 shows data for a small peptide that forms visible,
threadlike particles. The prominent peak near a radius of
Figure 10. Effects of incubation temperature on dissociation of
the aggregates in a thawed sample of Protein X over 24 hours, as
measured by the improved SEC method.
Figure 11. Example of a hydrodynamic radius distribution
derived from DLS data. The plot is a histogram of scattering
intensity (fraction of total scattering) as a function of
hydrodynamic radius.
Figure 12. Hydrodynamic radius distribution for a small peptide
that tends to form visible threadlike particles.
8. The AAPS Journal 2006; 8 (3) Article 65 (http://www.aapsj.org).
E571
100 nm is a precursor to those particles (its presence corre-
lates with manufacturing lots that eventually form the visi-
ble threads). Even though this peak represents over 90% of
the scattering intensity, its estimated weight fraction is only
0.002%. In several other cases we similarly have found crit-
ical precursors of visible particulates that had radii in the
range from approximately 20 to approximately 400 nm. In
2 cases that I can disclose we have been able to trace the
formation of those precursors to damage that occurred at
specific manufacturing steps (the viral clearance filter in
one case, a specific pump in another).
Some Drawbacks of DLS
On the other hand, DLS does have some important
weaknesses that should be mentioned. The first is the low
resolution. In general, 2 species are not resolved as separate
peaks unless their radii differ by a factor of approximately 2
(a factor of ~8 in molecular mass). Thus, DLS is usually a
poor tool for studying small oligomers; its best applications
are for very large aggregates. A second weakness is that
conversion of the intensity distribution (what the instrument
directly measures) into fractions by weight is subject to
many assumptions and sources of error; it is common to see
variations in weight fraction of a factor of 2 or more among
different aliquots of the same stock. Many useful applica-
tions of DLS, such as comparison of different formulations
or “good” versus “bad” lots, are fairly qualitative so this
relatively poor quantitation of weight fractions is fortu-
nately often not a significant problem. Last, because DLS
does not distinguish the chemical nature of the scattering
species, it is not always easy to tell whether large particles
are really product aggregates or some other type of contam-
inating particle.
CAN SEC BE REPLACED?
This discussion and the real-world examples have pointed
out several drawbacks and issues with SEC, and the regula-
tory agencies are well aware of these concerns. Nonetheless
it seems likely that SEC will continue to be the workhorse
tool for measuring aggregation for quite some time. The
alternative technologies discussed here still have one or
more of these major drawbacks: (1) they are not sufficiently
robust and easy to use to validate for lot release, (2) they
have low throughput, (3) they require expensive equipment
and highly trained personnel, and (4) the software may be
very far from being 21 CFR part 11 compliant.
What certainly can be done today, however, is to use these
alternate methods to help determine whether SEC is telling
the whole story. Doing such cross-validation usually does
not require a great deal of time or expense. When it does
appear the SEC method is missing important species, these
alternate methods can help guide the development of an
improved SEC method.
ACKNOWLEDGMENTS
I thank Vaxgen, Serono, and Integrity Biosolution for per-
mission to release data used in this manuscript.
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