HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMAT...Dr. Ravi Sankar
GASSCHROMATOGRAPHY[GC], ADVANCED STUDY OF THE FOLLOWING AND THEIR APPLICATIONS, INTRODUCTION, THEORY, COLUMN OPERATION,INSTRUMENTATION AND DETECTION,APPLICATIONS AND ADVANTAGES OF GC,PRINCIPLE OF SEPARATION IN GC, HOW GC MECHINE WORKS? COLUMN, DETECTORS.
BY P.RAVISANKAR, VIGNAN PHARMACY COLLEGE, VADLAMUDI, GUNTUR, A.P, INDIA.
Starch is found in corn, wheat ,potatoes and some other plant.Plastic packaging materials perform an important role in the food industry due to their durability, lightness, and flexibility which ceramics and metals cannot provide
Introduction to biopolymers,
Biocompatible and biodegradable polymers,
Applications of biopolymers,
Biopolymers used in advanced drug delivery systems-
Cellulose and its derivatives,
chitosan,
PLGA,
Polyanhydride,
polycaprolactone.
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMAT...Dr. Ravi Sankar
GASSCHROMATOGRAPHY[GC], ADVANCED STUDY OF THE FOLLOWING AND THEIR APPLICATIONS, INTRODUCTION, THEORY, COLUMN OPERATION,INSTRUMENTATION AND DETECTION,APPLICATIONS AND ADVANTAGES OF GC,PRINCIPLE OF SEPARATION IN GC, HOW GC MECHINE WORKS? COLUMN, DETECTORS.
BY P.RAVISANKAR, VIGNAN PHARMACY COLLEGE, VADLAMUDI, GUNTUR, A.P, INDIA.
Starch is found in corn, wheat ,potatoes and some other plant.Plastic packaging materials perform an important role in the food industry due to their durability, lightness, and flexibility which ceramics and metals cannot provide
Introduction to biopolymers,
Biocompatible and biodegradable polymers,
Applications of biopolymers,
Biopolymers used in advanced drug delivery systems-
Cellulose and its derivatives,
chitosan,
PLGA,
Polyanhydride,
polycaprolactone.
A biopharmaceutical, also known as a biological medical product, biological, or biologic, is any pharmaceutical drug product manufactured in, extracted from, or semi synthesized from biological sources are called protein drugs.
Irreversible protein aggregation is problematic in the biotechnology industry, where aggregation is encountered throughout the lifetime of a therapeutic protein, including during refolding, purification, sterilization, shipping, and storage processes. The purpose of the current review is to provide a fundamental understanding of the mechanisms by which proteins aggregate and by which varying solution conditions, such as temperature, pH, salt type, salt concentration, cosolutes, preservatives, and surfactants, affect this process.
Microbial Biotransformation of Pesticides(xenobiotics).pptxAliya Fathima Ilyas
* Biotransformation is the chemical modification made by an organism on a chemical compound, often associated with change in pharmacologic and toxicologic activity.
Introduction to Medicinal Chemistry, History and development of medicinal chemistry, Physicochemical properties in relation to biological action Ionization, Solubility, Partition Coefficient, Hydrogen bonding, Protein binding, Chelation, Bioisosterism, Optical and Geometrical isomerism, Drug metabolism Drug metabolism principles- Phase I and Phase II. Factors affecting drug metabolism including stereo chemical aspects
This presentation intends to offer the basic features of plant metabolism along with the different types of mechanisms to regulate and control the metabolic pathways.
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.
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.
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
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
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.
- 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
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.
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...
Aggregation Analysis of Therapeutic Proteins, Part 1: General Aspects and Techniques for Assessment
1. M
any proteins are marginally
stable in solution,
undergoing conformational
changes due to various
stresses during purification, processing
and storage. Such stresses can include
elevated temperature, shear strain,
surface adsorption, and high protein
concentration (1). Structurally altered
proteins have a strong tendency to
aggregate, often leading to eventual
precipitation (2). Irreversible
aggregation is a major problem for
long-term storage stability of
therapeutic proteins and for their
shipping and handling.
Aggregation problems have been
implicated in adverse reactions and
other safety issues since the beginning
of clinical applications of protein
pharmaceuticals. Immunoglobulin
aggregates have long been known to
cause anaphylactoid reactions (3, 4). In
recent years, a serious concern about
aggregation was raised by an upsurge in
incidents of pure red-cell aplasia
(PRCA) in patients receiving
recombinant erythropoietin, although it
appears that aggregation is not involved
in PRCA (5, 6). To minimize such risks
from therapeutic proteins in clinical
applications, formulations must be
optimized to reduce aggregation during
storage, handling, and shipping (7).
Size-exclusion chromatography
(SEC) has been a workhorse for
detecting and quantifying protein
aggregation (8, 9). But SEC is often
questioned because of possible loss of
proteins (soluble aggregates, in
particular) by their nonspecific
binding to the columns (10). Native
gels have also been used to observe
protein aggregation, but only
qualitatively. Column-free techniques
such as analytical ultracentrifugation
(AUC), field-flow fractionation
(FFF), and dynamic light scattering
(DLS) now find increasing application
in aggregation analysis. Here, we
review advantages and disadvantages
of various techniques for assessing
protein aggregation. In part 1, we
discuss general aspects of protein
aggregation and present two
techniques mentioned above: native
gel electrophoresis and SEC.
MECHANISMS OF AGGREGATION
Mechanisms of protein aggregation
are still not fully understood, despite
the fact that aggregation is a major
problem in therapeutic-protein
development. One plausible
mechanism is that aggregation is
driven or catalyzed by the presence of
a small amount of a contaminant.
That contaminant could be a damaged
form of the protein product itself, host
cell proteins, or even nonprotein
materials such as leachates or silica
particles (Figure 1A, pathway 1).
If the contaminant is the damaged
protein, then its aggregation may lead
to soluble oligomers, which become
larger aggregates, visible particulates,
or insoluble precipitates. Such soluble
oligomers, host-cell contaminants, or
nonprotein materials may serve as a
nucleus onto which native proteins
assemble and are incorporated into
larger aggregates (Figure 1A, pathway
2) (11). Damaged forms of a protein
product can arise from chemical
modification (such as oxidation or
deamidation) and from
conformationally damaged forms
arising from thermal stress, shear, or
surface-induced denaturation.
Minimizing protein aggregation from
this mechanism requires both
chemical and physical homogeneity;
that is, chemically modified or
conformationally altered proteins must
be removed from the final product.
A second mechanism begins with
partial unfolding of the native protein
Aggregation Analysis of
Therapeutic Proteins, Part 1
General Aspects and Techniques for Assessment
Tsutomu Arakawa, John S. Philo, Daisuke Ejima, Kouhei Tsumoto, and Fumio Arisaka
PRODUCT FOCUS: PROTEINS
PROCESS FOCUS: ANALYSIS AND
DOWNSTREAM PROCESSING
WHO SHOULD READ: QA/QC, PROCESS
DEVELOPMENT, MANUFACTURING, AND
ANALYTICAL PERSONNEL
KEYWORDS: ELECTROPHORESIS,
SIZE-EXCLUSION CHROMATOGRAPHY,
AGGREGATION
LEVEL: INTERMEDIATE
ANDREI TCHERNOV (WWW.ISTOCKPHOTO.COM)
Reprinted with permission from BioProcess International 4(10):42-43 (November 2006)
2. during storage (Figure 1A, pathway 2).
Protein conformation is not rigid —
the structure fluctuates around the
time-averaged native structure to
different extents depending on
environmental conditions. Some
partially or fully unfolded protein
molecules are always present at
equilibrium in all protein solutions,
but most such molecules simply refold
to their native structure. However,
those unfolded proteins may instead
aggregate with other such molecules
or may be incorporated into an
existing aggregate nucleus, eventually
to form larger aggregates as described
above. Factors such as elevated
temperature, shaking (shear and air-
liquid interface stress), surface
adsorption, and other physical or
chemical stresses may facilitate partial
unfolding.
A third aggregation mechanism is
reversible self-association of the native
protein to form oligomers. The content
of such reversible aggregates will
change with total protein
concentration, according to the law of
mass action. The tendency of different
proteins to reversibly associate is highly
variable, and the strength of that
association typically varies significantly
with solvent conditions such as pH and
ionic strength. In principle, these
reversible oligomers will dissociate
completely as the therapeutic protein
gets highly diluted after delivery in
vivo; consequently, they are somewhat
less of a concern than irreversible
aggregates. However, such reversible
oligomers often eventually become
irreversible (they are a first step along a
pathway to irreversible aggregation).
Thus, preventing accumulation of
irreversible aggregates may require
minimizing the reversible association.
Further, reversible self-association can
significantly alter important
pharmaceutical properties of product
solutions such as solution viscosity (12).
Detection of reversible aggregates
can be especially challenging. One
reason is simply that such aggregates
can dissociate from dilution during
the measurement process. A second,
less obvious reason is that the results
of any analysis method involving
separation may depend on the kinetic
rates of the reversible association–
dissociation reactions as well as the
equilibrium constants.
One consequence of the
complexities of aggregate formation
processes is the difficulty of linking
the effect (presence of aggregates) to
its underlying cause, particularly
because the key damage may occur at a
time or place quite separated from the
observed consequence. One example
arises during large-scale production of
therapeutic monoclonal antibodies
(MAbs), a key step in successful
product development. Acid stability
plays a major role in aggregation of
MAbs because their purification
process typically involves both low-pH
elution from protein-A affinity
columns and acid-treatment for viral
inactivation (Figure 1B) (13, 14).
Low-pH exposure of MAbs results
in small but significant
conformational changes that depend
on the pH, temperature, and solvent
composition. Although such partially
unfolded MAbs may not aggregate at
low pH, they often do aggregate
during subsequent manufacturing
steps involving changes in pH or ionic
strength (15). A larger conformational
change at low pH generally leads to
more aggregates upon pH increase.
Typically, aggregate formation from
the low-pH structure is not a fast
process, but it occurs slowly from
association of damaged monomers (as
depicted in Figure 1B) that have not
returned to their fully native structure.
Indeed, this and other types of
aggregation problems may not become
apparent until months after
manufacturing a particular lot of
protein or until later stages of product
development. Regardless of the
mechanism of aggregation, preventing
aggregation problems requires
sensitive and reliable technologies for
quantitative determination of
aggregate content and aggregate
characteristics.
AGGREGATION ANALYSIS
Aggregation analysis of proteins can
be formally classified into three
experimental modes. As shown in
Figure 2 (top panel), the first mode is
the most conventional approach: A
small volume of sample is applied to a
separation medium and forms a band
or zone. As the band migrates through
the medium, the proteins separate
according to differences in size,
electrophoretic charge, or mass. Gel
electrophoresis, SEC, FFF, and the
seldom-used band sedimentation
belong to this class. The movement of
the band or zone is monitored using
absorbance or refractive index
detection.
In the second mode of analysis,
initially the sample uniformly fills a
measurement cell (middle panel).
When an electrical or centrifugal
Figure 1: Pathway of protein aggregation
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3. driving force is then applied, the
protein moves along the applied field,
leaving protein-depleted solvent,
which creates a boundary between
protein-free and protein-containing
solution phases. The movement of this
boundary over time is measured. This
mode of separation is used in
sedimentation-velocity and moving-
boundary electrophoresis (an
electrophoresis originally developed by
Tiselius) (16).
The third category is a
measurement of particle size with no
physical separation (bottom panel). It
is termed correlation spectroscopy and
measures fluctuation of particles in
solution due to Brownian motion
(diffusion coefficients). Fluctuations
of scattered light and of fluorescence
intensity have been used. The most
widely used method in this category is
dynamic light scattering.
Native Gel Electrophoresis: Native
gel electrophoresis is a convenient way
to observe aggregation. For native
gels, sodium dodecyl sulfate (SDS) is
not used. The proteins retain their
native conformation, and their
mobility is governed by the ratio of
electric charge to hydrodynamic
friction. Hence, native gels neither
denature nor dissociate proteins.
Acidic proteins, which have isoelectric
points below 8, can be run in the
regular Tris-glycine Laemmli system.
For basic proteins, native gels are more
problematic and require optimization
of conditions, including the type of
buffer and gel, the pH, and the length
of electrophoretic run. Such gels are
run with the polarity reversed from
that of standard SDS gels and native
gels for acidic proteins. Because basic
proteins are positively charged, they
migrate toward the cathode as shown
in Figure 3 (far left panel). For acidic
proteins or on SDS-PAGE, proteins
are negatively charged and migrate
toward the anode.
Electrophoretic mobility of proteins
in the native gel depends on both the
electric charge and the hydrodynamic
size. It therefore gives information on
aggregation, provided that the charged
state of the oligomers or aggregates is
identical to that of the monomer.
Figure 3 shows native gel analysis of
Figure 3: Native gel electrophoresis of EPO — A, heated at 79 ºC with time in 20 mM phosphate; B,
heated at 79 ºC for 70 min in different buffers at 20 mM; C, heated at 79 ºC for 70 min as a function of
phosphate concentration; D, heated at 79 ºC for 70 min as a function of citrate concentration
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Figure 2: Classification of various techniques to analyze protein aggregation
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4. erythropoietin (EPO) that was
expressed in mammalian cells (CHO
cells) and hence is heavily
glycosylated. Aggregation of EPO was
induced by high-temperature
treatment (heat stress). The sample in
Figure 3A was heated at 79 °C in
20 mM phosphate for various times
and brought to room temperature for
the native gel analysis. Only a single
band is observed in the unheated
control (lane 1), suggesting that the
EPO preparation contains no
aggregates. However, a native gel with
Coomassie blue staining would not
detect 1–2% of small oligomers,
reflecting the sensitivity limit of such
staining. No change was seen in the
mobility after heating at 79 °C for 5–
15 minutes (lanes 2 and 3), indicating
no apparent formation of aggregates
within the limit of Coomassie blue
staining.
Because EPO is completely
unfolded at 79 °C (well above the
melting temperature of EPO) within a
few minutes, the observed full
recovery of the native protein indicates
that EPO readily regains the native
structure upon cooling (17, 18). After
30 minutes of heating (lane 4), a small
amount of low mobility band
corresponding to dimers is observed,
as indicated by the arrow (Figure 3A).
As the incubation time is increased,
more dimers and oligomers are formed
and the monomer decreases (lanes 5
and 6). The observed formation of
dimers and oligomers indicates that a
prolonged incubation causes unfolding
and consequent aggregation of EPO,
which cannot be reversed by low
temperature, an aggregation pathway
described in Figure 1A (pathway 2).
The formation of dimers, some of
which are covalently linked by
disulfide bonds, and higher oligomers
has been observed upon incubation at
elevated temperatures (data not
shown; see also 19). There is no dimer
formation when heated at 65 °C up to
90 minutes (data not shown), which is
still above the melting temperature of
EPO, indicating again that EPO
readily refolds to monomer.
We tested other buffers (citrate,
TrisHCl, histidine, and glycine) all at
20 mM for 70 minutes for their effects
on the reversibility of thermal
unfolding at 79 °C. The results in
Figure 3B show that these buffers have
no effect on the native protein (before
heating), whereas TrisHCl, histidine,
and glycine buffers gave a nearly
complete protection from aggregation.
Sedimentation velocity gave more
quantitative and detailed information
on the heat-induced aggregation of
EPO as will be described later (Part 2).
Next, native gel analysis was
applied to EPO samples heat-stressed
in different concentrations of
phosphate (Figure 3C). It is evident
that there are more soluble oligomers
at intermediate phosphate
concentrations. However, because
native gels are only semiquantitative it
is not clear whether monomer content
is actually increasing at higher
phosphate concentration, e.g., 0.2 M
(last lane), which illustrate a major
drawback of this approach. Figure 3D
shows the effect of citrate
concentration. The amounts of soluble
oligomers and monomers reach a
plateau at intermediate citrate
concentrations, above which there
appears to be no further increase in
aggregates. These native gel results
indicate that EPO aggregation occurs
after thermal unfolding and depends
on solvent conditions. Although this
approach is not quantitative and does
not fully reveal the nature of the
aggregates, it is a useful screening tool
to find solvent conditions that may
reduce aggregation.
Although natural and CHO-
derived EPO are highly soluble in
aqueous buffer because of heavy
glycosylation, unglycosylated EPO is
prone to aggregation because of its
high content of nonpolar amino acids
(17, 19). Heat-induced aggregation of
EPO as observed above may therefore
be due to exposure of hydrophobic
regions of the protein.
Native gels of purified monoclonal
antibodies (MAb-a and MAb-b) show
a cluster of sharp bands due to
heterogeneous glycosylation, which
results in either charge or size
heterogeneity or both (Figure 4); each
band appears to correspond to a
homogeneous protein species. These
purified MAb samples contain about
3% aggregates as determined by
sedimentation velocity. Within the
detection limit of silver staining, no
bands are observed above these major
components, indicating little
aggregation, consistent with the
sedimentation velocity analysis.
Native gel analysis was also used to
analyze aggregates of these MAbs.
Aggregates were generated by
subjecting the purified MAbs to low
pH and high temperature (stressed
samples). The heat-stressed MAbs
thus obtained showed several bands
above the major bands (highlighted by
a blue bracket), indicating that heat-
treatment generated aggregates. Those
bands corresponding to aggregated
species are more diffuse than the
monomer band, suggesting that the
aggregate bands are heterogeneous.
Although native gels can separate
multiple forms of monomer antibodies
through heterogeneous glycosylation,
this technique is not as quantitative as
other techniques described here.
Size-Exclusion Chromatography
(SEC): As mentioned above, SEC is the
workhorse for aggregation analysis of
pharmaceutical proteins. Aggregation
analysis of pharmaceutical proteins
using this technique is an absolute
nearly always required for regulatory
approval. Figure 5 shows the
resolution of analytical SEC for
MAb-a using a G3000SWXL HPLC
column (Tosoh Biosciences) and an
elution solvent of 0.1 M phosphate,
pH 6.8, in the absence and presence of
0.2 M arginine. To generate
aggregates, the MAb sample was
subjected to low pH and heat
treatment (in the absence of arginine)
as described above. Panel A
corresponds with the SEC results of
the unstressed samples, whereas panel
B shows the results of the heat-
stressed samples. The nonstressed
MAb-a shows a sharp peak
corresponding with the monomeric
form of the sample. Only about 2%
total aggregates are present in the
sample.
It is well recognized, however, that
the SEC column matrix (stationary
phase) can nonspecifically bind proteins,
and this is especially true for aggregates
and, hence, makes aggregation analysis
5. ambiguous (10). We have shown that
adding arginine greatly reduces such
nonspecific binding (20). Figure 5A
(right panel) shows the effects of adding
0.2 M arginine to the elution solvent.
Although the total aggregate content is
similar (~2%), there is a clear increase in
one of the aggregate species (indicated
by arrow), suggesting that such
aggregates, although present in small
amount, are lost during SEC when
arginine is not present; there is no
decrease in the monomer recovery in
the presence of 0.2 M arginine. Figure
5B shows the SEC results for heat-
stressed MAb-a. It is evident that the
aggregate peaks are much larger than
those seen for the nonstressed MAb-a
(panel A), with the aggregate content
increasing to ~26% (from 2%).
However, the heat-stressed sample
actually contains many more aggregate
than the above value, as also shown in
Figure 5B (right panel). In the presence
of 0.2 M arginine, the aggregate peaks
are much larger, with the aggregate
content at 43%, indicating again that a
large number of aggregates are lost
during the SEC unless arginine is
present. It should be noted that the
observed increase in aggregate content
is not due to formation of aggregates by
arginine during SEC. First, the amount
of monomer eluting is not decreased by
the arginine. Second, in our limited
experience, arginine does not denature,
nor does it enhance aggregation for
several proteins tested (21).
Obviously, careful monitoring of
total sample recovery during
chromatography should reveal the
poor recovery of aggregates such as
that in Figure 5B and serve as a
warning that the SEC method is not
working properly. However, even very
careful monitoring of total recovery
may not reveal subtle differences such
as those shown in Figure 4A.
Figure 6 shows the SEC results for
MAb-b. The unstressed sample
contains only ~1% aggregates whether
the SEC is done in the absence or
presence of 0.2 M arginine (panel A).
Peaks corresponding to aggregates are
much larger for the stressed sample
(panel B): ~38% in the absence of
arginine and ~43% in the presence of
0.2 M arginine, indicating again that
arginine suppresses nonspecific
binding of aggregated antibodies,
leading to a higher recovery of the
aggregates. It appears that the
shoulder of the aggregate peak
(indicated by arrow) is higher in the
presence of arginine, suggesting that
larger aggregates, which elute earlier,
tend to stick to SEC columns in the
absence of arginine. Of course, it is
uncertain whether arginine completely
suppresses the loss of aggregates on
the column.
It is thus evident that inclusion of
arginine in the SEC mobile phase
suppresses protein adsorption to the
silica surface. This is most likely due
to preferential binding of arginine to
the unbound protein, which is in
equilibrium with the surface bound
form (22, 23). As in Figure 7, arginine
shifts the equilibrium binding of
antibodies toward the dissociated
state. Can arginine then dissociate the
bound proteins from the column? The
answer is no, at least for the MAb-a
tested. As depicted in Figure 7, the
protein molecules bound to the SEC
column surface often undergo
conformational changes, leading to
binding-elution hysteresis or
irreversible binding (24). Arginine is
incapable of dissociating these
stronger interactions.
Another important advantage of
arginine is that it reduces lot-to-lot
manufacturing variation of SEC
columns in aggregation analysis, as
illustrated in Figure 8. Three lots of
Superdex-75 (GE Healthcare, www.
chromatography.
amershambiosciences.com) show a
varying degree of aggregate content
for interleukin-6. An identical
antibody sample was applied to the
three different lots of a same column
type. As shown in panel A, little
aggregate is eluted from this column,
although the sample contains a
significant number of aggregates.
The column lot shown in panel C
showed a much larger aggregate peak
than the other two lots (panel A and
B), indicating that nonspecific
adsorption of proteins varies with
different lots. When 0.25 M arginine
was included, this variability
disappeared. As shown in three
panels, with arginine the aggregate
peaks are all sharp and similar in
size, indicating that arginine
suppresses nonspecific protein
adsorption, independent of the lots.
The ratio of the total aggregate
content measured in the absence or
presence of 0.25 M arginine is ~0 for
lot 0314065 (no distinct peak
observed in the absence of arginine),
0.37 for lot 0437127, and 0.95 for lot
0511099. That is, the last lot is least
sticky, at least for these aggregates.
Thus, inclusion of arginine in the
elution solvent eliminates the need to
reoptimize elution conditions when
changing to a new lot of columns.
The molecular weight of
aggregates can be determined using
an on-line light-scattering detector,
which measures the amount of light
scattered by the proteins. When
combined with the protein
concentration data from UV
absorbance or refractive index
detectors, the light scattering can
give the average molecular weight for
each peak eluting from an SEC
column. The molecular weight of the
aggregates relative to the monomer
indicates the stoichiometry of the
aggregates formed.
LOOKING AHEAD
As shown here, the mechanisms of
protein aggregation can introduce
problems. Several technologies can
be used to determine that level of
aggregation, among which are native
gel electrophoresis and SEC. In part
Figure 4: Native gel electrophoresis of MAb,
7.5 %T homogeneous gel (Phastgel); purified,
1 μg; heated, 2 μg; silver staining
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6. 2, we will introduce three other
technologies: sedimentation velocity,
FFF, and DLS, which are all free
from the column matrix.
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Figure 5: SEC analysis of MAb-a
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Figure 6: SEC analysis of MAb-b
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7. Corresponding author Tsutomu Arakawa
is president of Alliance Protein
Laboratories, Thousand Oaks, CA 91360,
tarakawa2@aol.com; John S. Philo is vice
president and director of biophysical
chemistry at Alliance Protein Laboratories;
Daisuke Ejima is a chief chemist at
AminoScience Laboratories, Ajinomoto
Co., Inc., Kawasaki, Kanagawa 210-8681,
Japan; Kouhei Tsumoto is an associate
professor at the Department of Medical
Genome Sciences, The University of Toyko,
Kashiwa 277-8562, Japan; and Fumio
Arisaka is an associate professor in the
Department of Molecular Bioprocessing,
Tokyo Institute of Technology, Yokohama
226-8501, Japan.
Figure 8: SEC analysis of aggregation of interleukin-6; an interleukin-6 monomer–dimer mixture
(13 μg) sample with 0.1 M Na phosphate buffer, pH 6.8, containing 0.25 M NaCl or arginine
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Figure 7: Non-specific protein adsorption to
surface
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