This document discusses methods for characterizing proteins and their aggregation using sedimentation velocity and sedimentation equilibrium. Sedimentation velocity can detect different molecular species and quantify aggregates. It is useful for assessing sample homogeneity and comparability between batches. Sedimentation equilibrium determines molecular mass and quaternary structure, identifying whether proteins exist as monomers, dimers, etc. Both techniques provide complementary information about protein conformation and self-association important for developing protein therapeutics.
Metabolomics is the newest hype in the 'omics' family. Although defined as highly important to recent biological research, the analytical science applied is far from acceptable for the majority of publications that appear nowadays. Take a look at our approach to tackle the metabolomics issue, which remains a new name for an old science.
The study and understanding of the structure and properties of styrene/vinylester (St/VE) and styrene/unsaturated polyester (St/UP) cross-linked thermosets has received technologic and scientific attention, because these resins are widely used as matrices in composites formulations, sharing advantages, such as low room temperature viscosity coupled with good mechanical properties and low cost, plus the added chemical resistance in the case of VE.
Metabolomics is the newest hype in the 'omics' family. Although defined as highly important to recent biological research, the analytical science applied is far from acceptable for the majority of publications that appear nowadays. Take a look at our approach to tackle the metabolomics issue, which remains a new name for an old science.
The study and understanding of the structure and properties of styrene/vinylester (St/VE) and styrene/unsaturated polyester (St/UP) cross-linked thermosets has received technologic and scientific attention, because these resins are widely used as matrices in composites formulations, sharing advantages, such as low room temperature viscosity coupled with good mechanical properties and low cost, plus the added chemical resistance in the case of VE.
METABOLOMICS is the systematic study of the small molecular metabolites in a cell, tissue, biofluid, or cell culture media that are the tangible result of cellular processes or responses to an environmental stress.
Atomic force spectroscopy, a technique derived from Atomic Force Microscopy (AFM), allowed us to distinguish nonspecific and specific interactions between the acetolactate synthase enzyme (ALS) and anti-atrazine antibody biomolecules and the herbicides imazaquin, metsulfuron-methyl and atrazine. The presence of specific interactions increased the adhesion force (Fadh) between the AFM tip and the herbicides, which made the modified tip a powerful biosensor. Increases of approximately 132% and 145% in the Fadh values were observed when a tip functionalized with ALS was used to detect imazaquin and metsulfuron-methyl, respectively. The presence of specific interactions between the atrazine and the anti-atrazine antibody also caused an increase in the Fadh values (approximately 175%) compared to those observed when using an unfunctionalized tip. The molecular modeling results obtained with the ALS enzyme suggest that the orientation of the biomolecule on the tip surface could be suitable for allowing interaction with the herbicides imazaquin and metsulfuron-methyl
Introduction
Proteins
Function Of Protein And Their Properties
Protein Isolation And Purification
Methods Of Cell Lysis
Steps Of Protein Characterisation:
Determination Of Protein Concentration
Biuret Reaction
Lowry (Folin-Lowry) Method
UV- Spectroscopy
Assessment Of Protein Purity
SDS -Phage
Immunoblot
Surface Charge Analysis
Isoelectro Focusing
Ion Exchange Chromatography
Size, Shape And Conformation Analysis
2d-Electrophorasis
X-Ray Crytalliography
Protein Structure and Sequence Analysis
Edman Sequencing
Conclusion
References
Metabolites have various functions, including fuel, structure, signaling, stimulatory and inhibitory effects on enzymes, catalytic activity of their own (usually as a cofactor to an enzyme), defense, and interactions with other organisms (e.g. pigments, odorants, and pheromones).
Metabolome refers to the complete set of chemical compounds involved in an organism's metabolism (such as metabolic intermediates, hormones and other signaling molecules, and secondary metabolites)
Metabolomics is the scientific study of chemical processes involving metabolites. Metabolomics is a relatively new member to the ‘-omics’ family of systems biology technologies.
Discovery of active site of vinblastine as application of nanotechnology in m...Nanomedicine Journal (NMJ)
Objective(s):
Vinblastine is antimitotic, anticancer medicine that disturbs normal microtubule formation and favours depolymerisation. Structural study and finding the active site of vinblastine are the targets of this research.
Materials and Methods:
Vinblastine was optimized in vacuum and then in different solvents by Density Functional Theory (DFT) method. Nuclear Magnetic Resonance (NMR) shift measurements were made in different solvents by various dielectric constants by Continuous Set of Gauge Transformations (CSGT).
Results:
The best structure and function of vinblastine was established. The conformational preferences may be attributed to stereoelectronic effects. The results showed that the structure of vinblastine is more stabile in water rather than the other media. The most active atoms of vinblastine were realized by various spectra of vinblastine in different media including vacuum and diverse solvents.
Conclusion:
Discovery of active site of vinblastine that could bind to tubulin to perform the antimitosis and anticancer effect in process of cell division was accomplished in this investigation. These data can be applicable to study the binding site of vinblastine-tubulin complex.
METABOLOMICS is the systematic study of the small molecular metabolites in a cell, tissue, biofluid, or cell culture media that are the tangible result of cellular processes or responses to an environmental stress.
Atomic force spectroscopy, a technique derived from Atomic Force Microscopy (AFM), allowed us to distinguish nonspecific and specific interactions between the acetolactate synthase enzyme (ALS) and anti-atrazine antibody biomolecules and the herbicides imazaquin, metsulfuron-methyl and atrazine. The presence of specific interactions increased the adhesion force (Fadh) between the AFM tip and the herbicides, which made the modified tip a powerful biosensor. Increases of approximately 132% and 145% in the Fadh values were observed when a tip functionalized with ALS was used to detect imazaquin and metsulfuron-methyl, respectively. The presence of specific interactions between the atrazine and the anti-atrazine antibody also caused an increase in the Fadh values (approximately 175%) compared to those observed when using an unfunctionalized tip. The molecular modeling results obtained with the ALS enzyme suggest that the orientation of the biomolecule on the tip surface could be suitable for allowing interaction with the herbicides imazaquin and metsulfuron-methyl
Introduction
Proteins
Function Of Protein And Their Properties
Protein Isolation And Purification
Methods Of Cell Lysis
Steps Of Protein Characterisation:
Determination Of Protein Concentration
Biuret Reaction
Lowry (Folin-Lowry) Method
UV- Spectroscopy
Assessment Of Protein Purity
SDS -Phage
Immunoblot
Surface Charge Analysis
Isoelectro Focusing
Ion Exchange Chromatography
Size, Shape And Conformation Analysis
2d-Electrophorasis
X-Ray Crytalliography
Protein Structure and Sequence Analysis
Edman Sequencing
Conclusion
References
Metabolites have various functions, including fuel, structure, signaling, stimulatory and inhibitory effects on enzymes, catalytic activity of their own (usually as a cofactor to an enzyme), defense, and interactions with other organisms (e.g. pigments, odorants, and pheromones).
Metabolome refers to the complete set of chemical compounds involved in an organism's metabolism (such as metabolic intermediates, hormones and other signaling molecules, and secondary metabolites)
Metabolomics is the scientific study of chemical processes involving metabolites. Metabolomics is a relatively new member to the ‘-omics’ family of systems biology technologies.
Discovery of active site of vinblastine as application of nanotechnology in m...Nanomedicine Journal (NMJ)
Objective(s):
Vinblastine is antimitotic, anticancer medicine that disturbs normal microtubule formation and favours depolymerisation. Structural study and finding the active site of vinblastine are the targets of this research.
Materials and Methods:
Vinblastine was optimized in vacuum and then in different solvents by Density Functional Theory (DFT) method. Nuclear Magnetic Resonance (NMR) shift measurements were made in different solvents by various dielectric constants by Continuous Set of Gauge Transformations (CSGT).
Results:
The best structure and function of vinblastine was established. The conformational preferences may be attributed to stereoelectronic effects. The results showed that the structure of vinblastine is more stabile in water rather than the other media. The most active atoms of vinblastine were realized by various spectra of vinblastine in different media including vacuum and diverse solvents.
Conclusion:
Discovery of active site of vinblastine that could bind to tubulin to perform the antimitosis and anticancer effect in process of cell division was accomplished in this investigation. These data can be applicable to study the binding site of vinblastine-tubulin complex.
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
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
Introduction.
Types of Protein – Protein Interaction.
Methods to investigate Protein – Protein Interaction.
Protein – Protein Interaction modulated by Chemical energy.
Two Hybrid Screening.
Overview of Protein – Protein Interaction analysis.
Biological effect of Protein – Protein interaction.
Conclusion.
Reference.
Prediction of the three dimensional structure of a given protein sequence i.e. target protein from the amino acid sequence of a homologous (template) protein for which an X-ray or NMR structure is available based on an alignment to one or more known protein structures
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
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Acute scrotum is a general term referring to an emergency condition affecting the contents or the wall of the scrotum.
There are a number of conditions that present acutely, predominantly with pain and/or swelling
A careful and detailed history and examination, and in some cases, investigations allow differentiation between these diagnoses. A prompt diagnosis is essential as the patient may require urgent surgical intervention
Testicular torsion refers to twisting of the spermatic cord, causing ischaemia of the testicle.
Testicular torsion results from inadequate fixation of the testis to the tunica vaginalis producing ischemia from reduced arterial inflow and venous outflow obstruction.
The prevalence of testicular torsion in adult patients hospitalized with acute scrotal pain is approximately 25 to 50 percent
The prostate is an exocrine gland of the male mammalian reproductive system
It is a walnut-sized gland that forms part of the male reproductive system and is located in front of the rectum and just below the urinary bladder
Function is to store and secrete a clear, slightly alkaline fluid that constitutes 10-30% of the volume of the seminal fluid that along with the spermatozoa, constitutes semen
A healthy human prostate measures (4cm-vertical, by 3cm-horizontal, 2cm ant-post ).
It surrounds the urethra just below the urinary bladder. It has anterior, median, posterior and two lateral lobes
It’s work is regulated by androgens which are responsible for male sex characteristics
Generalised disease of the prostate due to hormonal derangement which leads to non malignant enlargement of the gland (increase in the number of epithelial cells and stromal tissue)to cause compression of the urethra leading to symptoms (LUTS
263778731218 Abortion Clinic /Pills In Harare ,sisternakatoto
263778731218 Abortion Clinic /Pills In Harare ,ABORTION WOMEN’S CLINIC +27730423979 IN women clinic we believe that every woman should be able to make choices in her pregnancy. Our job is to provide compassionate care, safety,affordable and confidential services. That’s why we have won the trust from all generations of women all over the world. we use non surgical method(Abortion pills) to terminate…Dr.LISA +27730423979women Clinic is committed to providing the highest quality of obstetrical and gynecological care to women of all ages. Our dedicated staff aim to treat each patient and her health concerns with compassion and respect.Our dedicated group ABORTION WOMEN’S CLINIC +27730423979 IN women clinic we believe that every woman should be able to make choices in her pregnancy. Our job is to provide compassionate care, safety,affordable and confidential services. That’s why we have won the trust from all generations of women all over the world. we use non surgical method(Abortion pills) to terminate…Dr.LISA +27730423979women Clinic is committed to providing the highest quality of obstetrical and gynecological care to women of all ages. Our dedicated staff aim to treat each patient and her health concerns with compassion and respect.Our dedicated group of receptionists, nurses, and physicians have worked together as a teamof receptionists, nurses, and physicians have worked together as a team wwww.lisywomensclinic.co.za/
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
Dr Sujoy Dasgupta presented the study on "Couples presenting to the infertility clinic- Do they really have infertility? – The unexplored stories of non-consummation" in the 13th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2024) at Manila on 24 May, 2024.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
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.
- 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
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
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
Characterizing the aggregation and conformation of protein therapeutics
1. A major difference between protein-
based and small-molecule pharmaceuti-
cals is that the bioactivity of proteins is
strongly dependent on molecular confor-
mation. Although we are blessed with
good tools for characterizing the primary
covalent structure of proteins, such as
peptide mapping and mass spectrometry,
these tools cannot tell us whether the
protein is in the correct, folded structure
in solution. Proteins also participate in
noncovalent self-association (oligomeri-
zation) reactions. These association reac-
tions may be either desirable (for exam-
ple, the native functional state may be a
dimer) or undesirable (producing aggre-
gates, a common and often vexing degra-
dation pathway). Therefore, for any po-
tential protein therapeutic, tools are
needed to establish 1) the native, biolog-
ically active, state of association; 2)
whether degraded conformations such as aggre-
gates are present; and 3) whether the same pro-
tein conformation can be made reproducibly. Two
related methods, sedimentation velocity and sed-
imentation equilibrium, are excellent choices for
addressing these needs. Both are available with
the ProteomeLab Optima XL-A/XL-I (Beckman
Coulter, Fullerton, CA).
Sedimentation velocity
Sedimentation velocity is a separation method
that provides a powerful means of characterizing
the homogeneity of protein samples (homo-
geneity of conformation and/or solution molec-
ular mass), particularly for detecting and quan-
tifying irreversible or long-lived aggregates.
Molecules are separated on the basis of their
sedimentation coefficient, a molecular parame-
ter that increases with higher molecular mass,
but that also depends on molecular shape
(because hydrodynamic friction is shape de-
pendent). Sedimentation coefficients can be
measured with high precision, and thus provide
an efficient means of demonstrating that sam-
ples from different manufacturing lots, or mate-
rial from different purification processes, all
contain the same molecular conformation
(comparability protocols). Depending on con-
figuration, up to seven samples can be run
simultaneously, at typically 2–4 hr per run.
Figure 1 shows an example of detecting aggregates
in a monoclonal antibody sample. Modern meth-
ods of sedimentation velocity data analysis can
convert the raw data into this distribution of sedi-
mentation coefficients. Like a chromatogram, each
peak represents a different species (different sedi-
mentation coefficient), and the area under each
peak is proportional to the concentration. This
accelerated-stability sample was highly stressed,
producing many peaks from degradation products
in addition to the main peak, antibody monomer
(a normal heterotetramer of heavy and light
chains), which sediments at 6.4 Svedbergs (6.4 S).
A series of well-resolved peaks sedimenting faster
than the main peak represent aggregates. While
we cannot uniquely assign a mass to those ag-
gregate species based on only these data, it can
be shown that these peaks represent dimer,
trimer, etc., to heptamer (the hexamer and hep-
tamer peaks are too small to see without ex-
panding the scale). Additional slowly sediment-
ing peaks presumably represent antibody
fragments (possibly half-molecules and
free light- or heavy-chain).
The fact that all of these different species
are separated and resolved as individual
peaks indicates that these are long-lived
species (lifetimes comparable to or longer
than the separation time of ~2–3 hr).
Therefore, these are irreversible (or only
very slowly reversible) aggregates, rather
than rapidly reversible self-associated
oligomers. (A rapidly reversible associa-
tion process will usually produce a con-
centration-dependent shift in the peak
position, but the different oligomers will
generally not resolve as individual peaks.)
Comparability testing
An example of using sedimentation
velocity for comparability testing of two
manufacturing lots of monoclonal antibody is
shown in Figure 2. The good news from this result
is that both lots show good homogeneity (98.6%
main peak or better) and the main peak occurs at
the same sedimentation coefficient (the mean
value over the peak equals 6.339 S for lot 1 and
6.335 S for lot 2), which proves that the molecu-
lar conformation is the same. Indeed, one of the
benefits of this approach is that sedimentation
coefficients are absolutely calibrated (not relying
on molecular standards) and can be measured to a
precision of ±0.2% or better for comparisons
within the same run and ±0.5% run-to-run. The
bad news from this comparison, however, is that
the levels and types of aggregates in these two lots
appear to be somewhat different, as can be seen in
the graph inset (vertically expanded 100-fold).
Is this approach actually reliable for detecting
aggregate species such as those that are present at
levels of only a few tenths of a percent or less?
Experience shows that for minor peaks that are
near the large main peak (such as the ~10 S dimer
in these samples), the variability in area corre-
sponds to ±0.2–0.3% of the total, and there is
some sample-to-sample variation in the peak
positions. However, peaks that are well separated
22 / OCTOBER 2003 • AMERICAN BIOTECHNOLOGY LABORATORY
A P P L I C A T I O N N O T E
Characterizing the Aggregation and
Conformation of Protein Therapeutics
by John S. Philo
Figure 1 Sedimentation coefficient distribution for a highly stressed monoclonal
antibody sample.
2. from the main peak, such as the ~23 S
species in lot 2, can be reliably detected
down to levels of 0.05% or lower. Thus,
the differences in aggregate content and
distribution for these two samples are
indeed significant in comparison to the
reproducibility of the method.
Another important aspect of this method
is that samples can generally be run
directly in their formulation buffers.
There is also no potential irreversible
binding of aggregate species to a column
resin. In contrast, for size-exclusion (gel
filtration) chromatography (SEC), which
is often employed as an aggregation assay,
the elution buffer usually must be at quite
high ionic strength. Further, aggregate
species are often much more sticky than
the native state, and they are easily lost to
the column matrix. Because of this, and
to obtain good resolution and symmetric
peaks, chromatographers often add or-
ganic cosolvents and/or use strongly
acidic elution buffers. However, the use of
an elution buffer that is very different
from the formulation buffer, or even par-
tially denaturing, may drastically alter the
distribution of noncovalent aggregates
that was initially present.
Sedimentation
equilibrium
While the strong suit of sedimentation
velocity is molecular conformation
and the characterization of mixtures of
different species, sedimentation equi-
librium is a complementary tool the
strengths of which are measuring mo-
lecular mass in solution and studying
samples involved in rapidly reversible
binding equilibria (self-association or
binding between different macromole-
cules). In contrast to the strong separation
applied during sedimentation velocity experi-
ments, in sedimentation equilibrium, only a
very gentle force is applied, allowing the sample
to maintain thermodynamic equilibrium for its
binding interactions.
One major and important use of sedimentation
equilibrium is simply to identify whether the
native state of a protein in solution is monomer,
dimer, or some higher oligomer (determination
of quaternary structure). This may seem fairly
trivial, but often the state of association is im-
portant for biological function (e.g., a ligand
may need to be a dimer in order to dimerize its
cell-surface receptor); over the years the author
has seen a remarkable number of cases in which
such assignments were made incorrectly.
The need for this type of data often arises at the
two extreme ends of the development cycle. At
the discovery stage, new leads or targets have
usually been identified by proteomic and/or
genomic approaches, and often on the basis of
homology to other proteins known to be biolog-
ically or therapeutically significant. When the
interest in a particular protein is based largely
on homology, it is important to confirm that
there is actually homology of solution
structure and conformation, including
the correct state of association.
One such example is a new homologue
of tumor necrosis factor (TNF). When
cloned and expressed in E. coli, this pro-
tein was assigned as a monomer based
on SEC, whereas a trimeric structure is
a hallmark of the TNF family. Hence, a
monomeric structure would suggest
either that this was not a true TNF
homologue, or possibly that the protein
had not been correctly refolded from
inclusion bodies. However, as shown in
Figure 3, sedimentation equilibrium eas-
ily showed that the protein is indeed a
trimer in solution.
When a protein therapeutic enters clin-
ical development, a measurement of so-
lution molecular mass is usually in
cluded as part of the basic characteriza-
tion package. Precisely because methods
such as SEC do not always provide the
correct solution mass, the regulatory
agencies prefer a more robust method
that is independent of molecular shape.
Thus, sedimentation equilibrium data
are increasingly used to provide this
basic characterization information.
Such data can also be useful if compara-
bility studies are needed to support
changes in purification or formulation
that may be needed during clinical
development or for postapproval manu-
facturing changes. Depending on the
configuration, up to 28 samples can be
run simultaneously.
Some proteins may not exist in essen-
tially a single state of association, but
instead may be present in reversible
association equilibrium between two or
more association states at the protein concen-
tration and solution conditions used for the for-
mulation. The characterization of such reversi-
ble interactions is a particular strength of
sedimentation equilibrium, but space limita-
tions prevent examples and details of those
applications to be given here.
Formation of protein
aggregates at high
concentrations
The fact that many new protein therapeutics,
A P P L I C A T I O N N O T E
Figure 2 Comparability of conformation and aggregation for two different manufacturing
lots of a monoclonal antibody.
Figure 3 Sedimentation equilibrium data for a sequence homologue of tumor necrosis
factor-α, part of a family of proteins that are usually trimers. In this form of plot, a single
species gives a straight line with a slope proportional to solution mass. The blue line shows the
slope predicted for a monomer, while the red is for a trimer. Clearly, this protein is indeed a
trimer, as expected.
24 / OCTOBER 2003 • AMERICAN BIOTECHNOLOGY LABORATORY
3. especially monoclonal antibodies, are being for-
mulated at high protein concentrations (10–50
mg/mL or higher) has heightened concerns
about aggregation and the potential immuno-
genicity or pharmacokinetic changes that may
result. This concern is also creating consider-
able confusion. One source of confusion is the
term “aggregate” itself. Proteins can associate to
form either reversible or irreversible oligomers.
The reversible oligomers are held together by
noncovalent bonds only; the irreversible ones
may be linked covalently (e.g., by disulfide
bonds) or noncovalently. Further, as illustrated
in Figure 4, reversible oligomers are generally
precursors to the irreversible ones; therefore
pushing up the protein concentration can drive
formation of both types.
Some scientists term all oligomers “aggregates,”
including the native state of the TNF homo-
logue discussed above; some reserve the label for
irreversible cases only, and others adopt inter-
mediate definitions. Adding further to the mis-
understanding is the fact that some analytical
methods may detect only a subset of the various
types (or sizes) of oligomers, either because they
are strongly dissociating for noncovalent inter-
actions (like sodium dodecyl sulfate-polyacry-
lamide gel electrophoresis [SDS-PAGE]), or
because the measurement involves a large dilu-
tion that dissociates weak rapidly reversible
interactions (like SEC). What is perhaps least
appreciated and most confusing, however, is the
fact that reversible association–dissociation
reactions can sometimes have surprisingly slow
kinetics, such that it may take hours or even
days to reestablish mass-action equilibrium after
a change in concentration, pH, or temperature.
Thus, whether oligomers are detected by a given
separation method can depend on the time
scale of the separation relative to the kinetics of
association–dissociation, and when the kinetics
are slow the results may depend strongly on the
sample preparation history.
Are all types and sizes of oligomers of equal con-
cern? With regard to size, there appear to be few
quantitative data about the relative effects of
dimers, trimers, and tetramers, etc., but very
large aggregates are a particular concern for
immunogenicity. Is there cause for concern if,
for example, a protein only forms dimers at con-
centrations above 10 mg/mL? If these dimers
dissociate very rapidly (a few minutes or less) as
the protein is diluted in vivo, then it seems
unlikely they will have any effect on safety or
efficacy. On the other hand, oligomers that are
irreversible or that persist for several hours
clearly could potentially alter pharmacokinet-
ics, potency, and/or immunogenicity. Thus, the
lifetime of an oligomer or aggregate can be an
important parameter both for biological conse-
quences and for selecting and evaluating appro-
priate analytical methods. Sedimentation equi-
librium can detect all associated states, even
very rapidly reversible ones, whereas, as noted
above, for sedimentation velocity only the long-
lived species are resolved as separate species.
With regard to directly measuring the state of
association for samples at concentrations >10
mg/mL, unfortunately, even when one can make
the measurements, the interpretation of all phys-
ical methods (light scattering, sedimentation,
osmotic pressure, viscosity, etc.) is strongly com-
promised by the strong solution nonideality
(molecular crowding) effects. These effects, and
methods for high-concentration studies, are
complex topics well beyond the scope of this
article. However, one protocol that has been
found to be very useful for such samples is to
directly measure the long-lived aggregates by
diluting the samples down to ≤1 mg/mL and
immediately running sedimentation velocity. A
comparison of such data to samples equilibrated
at low concentration for several days allows dis-
crimination of the very slowly dissociating
aggregates, and dilution into phosphate-buffered
saline can be used to more closely mimic the
dilution that will happen in vivo.
Summary
The above techniques provide a complementary
set of true solution methods for characterizing
protein conformation, heterogeneity, state of
association, aggregation, and the strength of
solution binding interactions. Sedimentation
velocity is uniquely able to provide a sensitive
and quantitative way to demonstrate compara-
bility of molecular conformation, while sedi-
mentation equilibrium is widely considered the
“gold standard” for solution molecular mass.
These methods are based on simple physical
principles and do not rely on so-called standard
proteins for calibration. Indeed, their absolute
calibration even allows valid comparisons
between data collected years apart.
Dr. Philo is the Director of Biophysical Chemistry, Alliance
Protein Laboratories, 3957 Corte Cancion, Thousand Oaks,
CA 91360, U.S.A.; tel.: 805-388-1074; fax: 805-388-
7252; e-mail: jphilo@ap-lab.com.
A P P L I C A T I O N N O T E
Figure 4 Typical pathways for formation of protein oligomers
and aggregates. As noted, the rates of association/dissociation can
be remarkably slow (hours to days), obscuring the distinction
between reversible and irreversible events. The transition from
reversible to irreversible oligomers may, of course, occur at an ear-
lier stage (e.g., a disulfide-linked dimer). Not illustrated is the fact
that the initial association may be preceded by a partial unfolding of
the native monomer, or that association may be strongly promoted
by chemical degradation pathways (deamidation, oxidation).
26 / OCTOBER 2003 • AMERICAN BIOTECHNOLOGY LABORATORY