text:
**Featured in the Year's Best U.S Cabernet and Blends 2014 Issue** From a vineyard planted at 2,600 feet on the volcanic slopes above Clear Lake, this is a sophisticated cabernet, the fruit as deep and dark as blackberry juice, rich yet clean and spicy, laced with mountain aromas of pine straw and dry brush. Firm and almost formidable in structure, it opens into savory layers, lengthened by refreshing acidity.
(12/ 2014)
Big and rich, this needs time or a great steak to bring out its best. The color is very dark to the rim. Aromas are closed but the flavors compare to tart blackberry and black cherry with smoky accents. It has density, a layered texture that is tannic but not harsh, and a reserved power that should come out with time.
(2/ 2015)
A few interesting points concerning this wine: Obsidian Ridge, founded by winemaker Michael Blaise Terrien, is a member of Tricycle Wine Co., which exclusively uses their own Kádár Hungarian oak barrels. And while this Cabernet, blended with a bit of Cabernet Franc and Syrah, hails from the cool and rainy 2011 vintage, the high elevations from 2,300 feet in Lake County afforded the grapes better ripening conditions than existed during the season, for example, on the valley floor in Napa. The winemaker's note: "Slightly dusty and herbal on the nose, this Cabernet Sauvignon shows a wonderful similarity to classic Bordeaux blends from great vintages. These refined aromas are a prelude to the silky feast of ripe plums and black cherry flavors that fan out across the palate. A soft touch of vanilla adds to the allure of this polished offering. The unique vintage conditions helped shape what may be our most balanced vintage yet."
Below is a listing of the wine review scores we frequently reference in our notes. While these sources remain an excellent source of information, we always recommend that you trust your own palate. Scores in these publications often represent the personal taste of an individual critic, which may not always coincide with your own. We have a huge team of excellent wine professionals that can match wines you've had in the past with new arrivals that meet your flavor profile.
The item you have chosen is in stock, and has inventory in at least one of our retail
stores. Below is the current quantity on hand information for this product within our database. It is never more than five minutes old. Additionally, our shopping cart looks at real time inventory so when you add an item to you cart we will do an immediate check of available inventory and alert you if there are any issues.
2012 Von Strasser "Rudy" Napa Valley Cabernet Sauvignon
2012 Textbook Napa Valley Cabernet Sauvignon
2013 McManis California Cabernet Sauvignon
2012 Lewis Cellars "Reserve" Napa Valley Cabernet Sauvignon
2012 Larkmead Napa Valley Cabernet Sauvignon


text:
Protecting Your Tablet From a Fall From Space 130
from the nobody-can-hear-your-ipad-fall dept.
We've improved Slashdot's video section; now you can view our video interviews, product close-ups and site visits with all the usual Slashdot options to comment, share, etc. No more walled garden! It's a work in progress -- we hope you'll check it out (Learn more about the recent updates).
Appeared on Fark a couple days ago, with the comment that the (unprotected) camera they used to document the flight and fall also survived. So...
Per the Apple iPad 2 spec: Nonoperating temperature: -4 to 113 F (-20 to 45 C)
We didn't get a *real* good look at the display post-flight, but it seems the system was still usable after a cold soak down around at -23 F. Ok, so it wasn't that far out of spec, the system probably enjoy some solar heating, and it was a *dry* cold.
I can't quite explain why, but this is the most amazing thing I've since on slashdot in quite some time. Something about that little lonely iPad going all the way up there... my colleagues were just gathered around my computer and everyone exclaimed aloud.
The remains of the balloon are enough of a parachute, and the placement of the camera and mount can help it fall level as well.
After any salary raise, you will have less money at the end of the month than you did before.

subset: commoncrawl


text:
Crystal by MEElectronics In-Ear Headphones with Microphone Made with Swarovski® Crystals (Gold)
"When MEE was describing their new iems to me this is what they said: "... these earphones heavily emphasize comfort and appearance, they have nicely balanced sound that is less bassy compared to our M-series in-ears... We wanted to create an audio accessory women will actually want to wear – something that not only provides an exceptional listening experience, but is small and..." --indieman
Etymotic Research ER6i Isolator In-Ear Earphones (White)
" These were my first real earphones, and I bought them entirely because they'd been spreading like a plague on a forum I used. Upon first hearing them I was disappointed, but over time they grew on me, and until upgrading to the ER4, they were a firm favourite. Some of my friends couldn't get the fuss, nor understand why anybody would spend a hundred bucks on iPod earphones, but then again..." --BBBS
Etymotic Research ER6 Isolator In-Ear Earphones (Black)
" These were my first in-ears, and since owning them I've had the Ultimate Ears Triple Fi and the Klipsch Image S3s. I'll be comparing the ER6s to these. I remember the Etymotic's sound being very warm and natural, absolutely never harsh sounding. You could throw it the hissiest, most cymbal-laden music and it would sound wonderfully dark. It really had a..." --ledhed2222
Etymotic Research HF5 Portable In-Ear Earphones
" First off let me preface by stating that I bought these on a whim. I had read many things about Etymotic's penchant for accurate reproduction. The initial impressions I got from these were a mixed bag. First off, I love the isolation factor, putting these on in the middle of a crowded communal area was like stepping into a library. I tried the various silicone..." --Exarch
MEElectronics M-Duo
"what there is to say ? soundstage ruins it all. it has nearly the same soundstage with the already trash earphones like the brainwavz m4, xiaomi piston etc. the bass is really there but i've to say, 125-250-500hz frequencies comes overly amplified, still not as koss kdx100 but it is enough to give one a headache. so you'll probably need to have a proper equalizer setting in your source to lower..." --vaziyetu
Etymotic Research ER4B MicroPro Binaural In-Ear Earphones
" Nothing, not even a full-sized headphone, comes close to ER-4B in terms of tonal accuracy(diffuse-field). How accurate is it? Its target response is almost same as the one from the most up-to date study on the eardrum response in a diffuse-field. Although this product is advertised for binaural recordings only, it is basically same as ER-4S with the lack of room attenuation at the high..." --udauda
MEElectronics M31 In-Ear Headphones
M31 In-Ear Headphone; Fun is what the M31 is all about, giving you a fun, punchy presentation with good space and enhanced bass. Providing warmth along with 5 color choices of the the cool two-tone housing, this earphone delivers! The Original M Series in-ear headphones have a fun sound with enhanced bass that doesn't intrude into the rest of the spectrum, giving you the bass you want...
MEElectronics EP-DD53P-BK-MEE M-Duo Dual Driver Enhanced Bass In-Ear Headphone
Dual drivers for better sound - The M-Duo is perfect for those who want to feel the bass. With a dedicated subwoofer and crossover, each driver reproduces a different part of the frequency spectrum, which ensures your bass kicks hard while the rest of the spectrum remains clean and clear. The M-Duo delivers all of the impact and dynamics in your music. Prevent interruptions and block external...
Etymotic ER-4P Portable In-Ear Earphones
" I'm surprised that no review has been written about those gems! The ER 4P/S is one of the best IEMs in the market value wise. They are made to last, they isolate like no other IEM and they have a unique flat frequency response which can be easily adjusted if you have a good equalizer or an integrated amp. The ER S will sound best when used with a powerful amp, it will provide an amazing sound..." --homeros8000
MEElectronics CW31 Wooden In-Ear Headphones for iPod, iPhone, MP3/CD/DVD Players (Wood)
" I used to own MEElecs. George Carlin said that he never was fine and dandy in same moment. I was. Now, I am sad to say that I was so careless to lose them in worst possible scenario. They were not in their pouch, in my backpack, but alone. In my pants pocket. With foolish illusion of safety. One moment they were in my jeans, next one they weren't, how- three months later..." --milan425


text:
Hide Property Details
Description from the owner
- 1 bedrooms
- 1 bathrooms
- Sleeps 1
- Apartment
Bedrooms: 1
Bathrooms: 1
Sleeps: 1
Type: Apt. / CondoFlet Vista Mare
Description from the owner
Apartment in Brazil, a town of Piedade, in the stadium of Pernambuco with sea view a must, 40 meters from the beach, close to pharmacy, supermarket, shopping center, doctor, public transport and all those services is also not listed property consists of bedroom, kitchen , bathroom, living room, garage, swimming pool, sauna, concierge, located on the 12th floor of 4th garage, total 16 floor, 220,000 real asking price of € 85,000
Appartamento in Brasile, località di Piedade, nello stadio di Pernambuco con vista mare imperdibile, 40 mt dalla spiaggia, vicino di farmacia, supermercato, shopping center, dottore, mezzi pubblici e di tutti quei servizi ...Read more
About the owner
About the owner
nicola v.
nicola v.
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Calendar updated 4 months ago
Overall ratingNone
Flet Vista Mare
Recife, State of Pernambuco, Brazil
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text:
- Description
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World's Largest Aftermarket Motorcycle Parts and Accessories Superstore!
Input the part number and quantity to add a part directly to your cart.
Everyday Low Price: $4.99
Gold Club Price: What's This? $4.49
J&P Part: 174-552
This product has an attached note. View Product Note
This product was proudly made in the USA
jdloycec Posted:
This line of vest extenders looks great and very good quality.
doa313 Posted:
Requires a browser that supports HTML5.
This Vest Extender designed to work on a Woman's vest only , the snaps are revered from a Men's style vest.
Call Us 24/7 1-800-318-4823


text:
FORT MEADE, Md. (AP) -- A firefight erupted Monday when two people in a car tried to ram a National Security Agency gate at Fort Meade, killing at least one person...
BOSTON (AP) � Prosecutors rested their case against Boston Marathon bomber Dzhokhar Tsarnaev on Monday after jurors in his federal death penalty trial saw gruesome autopsy photos and heard a medical examiner describe the devastating injuries suffered by an 8-year-old boy killed in the 2013 terror attack.
CHARLOTTE, N.C. � Gov. Pat McCrory isn't backing bills giving exemptions to court officials who decline to perform certain marriages and offering other religious protections to businesses.
HOUSTON (AP) � Like his Duke teammates sitting on either side of him at the podium, Quinn Cook had a strand of the cut-down net sticking out of his regional championship cap.
DUNN, N.C. � The state chapter of the NAACP and the Forward Together Moral Movement are holding their fourth and final "Moral Monday" accountability organizing meeting to discuss the record of Rep. David Lewis, who serves Harnett County.
LONDON (AP) -- A disgruntled worker shoots up a workplace. A student opens fire at a high school. A pilot crashes a planeload of people into a mountainside.There may never be a convincing explanation for such devastating acts of violence, but...
Mostly Clear, 42�
Sunny, 69�
Listen for the Question of the Morning beginning at 5:20 am each weekday morning on North Carolina's Morning News on NewsRadio 680 WPTF and WPTF.com.
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Download your free app by seaching iTunes for "WPTF" or click the link for the iTunes store.
Need to know what to do in your garden month-by-month? WPTF's Weekend Garden Mike Raley has all the details
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text:
Book your room at Hampton Inn & Suites Cleveland-Beachwood
King Non SmokingMax Occupancy: 2 guests (up to 1 child, 1 infant)
Two Queen BedsMax Occupancy: 4 guests (up to 3 children, 3 infants)
One King Bed, Non-Smoking, AccessibleMax Occupancy: 2 guests (up to 1 child, 1 infant)
Studio King Suite Non SmokingMax Occupancy: 5 guests (up to 4 children, 4 infants)
Studio One King Bed SmokingMax Occupancy: 5 guests (up to 4 children, 4 infants)
Suite Two Queen BedsMax Occupancy: 6 guests (up to 5 children, 5 infants)
Beachwood hotel in Orange with free breakfast and indoor pool
Located in Orange, this hotel is 1.8 mi (3 km) from Maltz Museum of Jewish Heritage and 2.2 mi (3.5 km) from The Temple Tifereth Israel. St Dominic Catholic Church and ThistleDown Racetrack are also within 6 mi (10 km).
Hotel Features
This hotel features an indoor pool, a fitness center, and a spa tub. Free buffet breakfast, free WiFi in public areas, and free self parking are also provided. Other amenities include a 24-hour business center, a meeting room, and dry cleaning.
Room Amenities
All 139 rooms offer free WiFi and free wired Internet, plus coffee makers and flat-screen TVs with cable channels. Free weekday newspapers, free local calls, and hair dryers are among the other amenities that guests will find.Information missing or incorrect? Tell us!
Hotel Amenities
Hotel Amenities
In addition to an indoor pool, Hampton Inn & Suites Cleveland-Beachwood provides a spa tub and a fitness center. A computer station is located on site and high-speed wireless Internet access is complimentary. This hotel offers access to a 24-hour business center and a meeting room. Guests can enjoy a complimentary breakfast. Event facilities measure 750 square feet (68 square meters). This Beachwood hotel also offers complimentary newspapers in the lobby, a fireplace in the lobby, and laundry facilities. Complimentary self parking is available on site.
- Computer station
- Free WiFi
- Indoor pool
- Spa tub
- Free newspapers in lobby
- Fitness facilities
- 24-hour front desk
- Express check-out
- Express check-in
- Safe-deposit box at front desk
- ATM/banking
- Laundry facilities
- Dry cleaning/laundry service
- Fireplace in lobby
- Television in lobby
- Free breakfast
- Number of floors - 6
- Elevator/lift
- Total number of rooms - 139
- Conference space size (meters) - 68
- Free self parking
- One meeting room
- 24-hour business center
- Conference space size (feet) - 750
Family Friendly Amenities
- Indoor pool
- Free Wi-Fi
- Rollaway/extra beds available
- Laundry facilities
- Cribs/infant beds (complimentary)
Available in all rooms: Free WiFi , Free wired high-speed Internet
Available in some public areas: Free WiFi
Free self parking
Room Amenities
- Free cribs/infant beds
- Rollaway/extra beds available
- Premium TV channels
- Hypo-allergenic bedding available
- Air conditioning
- Blackout drapes/curtains
- Climate control
- Free local calls
- Coffee/tea maker
- Daily housekeeping
- Free toiletries
- Hair dryer
- Shower/tub combination
- Iron/ironing board
- Desk
- Free weekday newspaper
- Private bathroom
- Connecting/adjoining rooms available
- Cable TV service
- Flat-panel TV
- Free WiFi
- Free wired high-speed Internet
Where to Eat
Guests are offered a complimentary hot/cold buffet breakfast.
Nearby Things to Do
An indoor pool and a spa tub are on site. Other recreational amenities include a fitness center.
Children under 18 years old are not allowed in the swimming pool, fitness facility, or spa tub without adult supervision.
To make requests for specific accessibility needs, complete the "Special or Accessibility Requests" section of "Trip Preferences" when you book your hotel.
- Accessibility equipment for the deaf
- Accessible bathroom
- Accessible path of travel
- Braille or raised signage
- Accessible parking
- In-room accessibility
- Roll-in shower
Hotel Policies
Check-in time starts at 3 PM
Minimum check-in age is 21
Check-out time is noon
Payment types
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
We should mention
The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.


text:
I Meant Wired Headset & Turn Beats Off then volume will be very low as compared to sense 4 when we turn beats off
Is it necessary to s off or should i just unlock bootloader & install sense 4 rom via recovery?
XDA Developers was founded by developers, for developers. It is now a valuable resource for people who want to make the most of their mobile devices, from customizing the look and feel to adding new functionality.
Are you a developer?
Copyright © xda-developers. Hosted by Leaseweb


text:
Which One's Beth?
Megan Mullally (Children's Hospital, Party Down, Will & Grace) and Stephanie Hunt (Friday Night Lights) return to Largo with their band, Nancy and Beth. Their band? How did that come to be? "I met Stephanie last summer when we did an indie movie in Austin together called Somebody Up There Likes Me," Mullally tells us. "She told me she played ukulele. On the last day there, she played ukulele and we sang together, and that was it." Nancy and Beth's "An Evening of Songs for Men and Women" was a hit last month, with special guests Nick Offerman (Mullally's hubby) and That Dog's Petra Haden joining them for what Mullally calls "Stephanie and I singing and dancing like magical, rainbow pony princesses." So what rock star does she most relate to? "I guess, Tommy Lee because of my huge schlong and the fact I, too, love to bone Pamela Anderson." Largo at the Coronet, 366 N. La Cienega Blvd.; Wed., July 25, 9 p.m.; $30. (310) 855-0350.
Wed., July 25, 9 p.m., 2012
Related Location
366 N. La Cienega Blvd.
Los Angeles, CA 90048
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text: I have lived my life around the globe and have shared in the hospitality of people throughout the world. These have been some of the best experiences of my life. I have the opportunity now to be on the hosting side of things and am eager to continue to have many great experiences with people, yet this time sharing my space with people from across the planet.


text:
News 16 - 29 of 29
Raw Video of Ray J Beating Fabolous SurfacesSeptember 22, 2011 12:09:41
50 Cent who allegedly stirred up the whole thing by encouraging Ray to confront Fabolous was present to break the fight.
Emmy Viewers Drop 8 Percent From Last Year, but Rise in Younger DemosSeptember 20, 2011 08:09:07
The Sunday (September 18) telecast drew 12.4 million audience since it faced a stiff competition with the airing of Sunday Night Football on NBC.
Jennifer Lopez Casts Spell on Men in Full 'Papi' Music VideoSeptember 20, 2011 08:09:21
The estranged wife of Marc Anthony drives everyman in town crazy the day after she eats a 'love cookie' given by a friend.
Robert Pattinson Showed Up at Jimmy Fallon's Emmy After PartySeptember 20, 2011 01:09:04
Not a figure seen at the Sunday ceremony, Pattinson skipped the formality to attend Fallon's casual bash at Trousdale.
Ray J Says He Beat Up Fabolous and Would Do It AgainSeptember 20, 2011 01:09:25
Disputing the reports that he had been taken over by the rapper during their fist fight, Ray J challenged Fab to show his face to the public.
Fabolous Sued for Hitting Bus Boy in a RestaurantFebruary 03, 2011 10:02:25
Octavious Butler filed a lawsuit in Atlanta's Fulton County Superior Court, alleging he was harassed by the rapper and his entourage while cleaning their table.
New Videos: Fabolous' 'Sheep Clothing', Eric Benet's 'Live Without You'February 02, 2011 03:02:11
'Wolves in Sheep Clothing' features black-and-white scenes of Fabolous rapping his verses, and 'Never Want to Live Without You' sees Eric Benet's wedding.
Fabolous Debuts 'You Be Killin Em' Music VideoDecember 14, 2010 01:12:51
The new music video features James Bondesque spy espionage theme and has a cameo from Amber Rose as an assassin.
Amber Rose and Fabolous Shot Down Rumors They're Expecting ChildOctober 07, 2010 02:10:56
While Rose laughed off the rumors and tweeted, 'The internet is 97 per cent wrong about me,' Fabolous insisted, 'Despite the rumor I didn't get anyone pregnant.'
Soulja Boy and Fabolous Call a TruceSeptember 10, 2010 09:09:43
The rappers who were fighting on Twitter had made peace, saying it was a misunderstood joke.
Fabolous Left Lyric-Filled Phone in EateryJuly 22, 2010 10:07:26
Luckily, the waiter of Morton's Steakhouse was able to catch up with him and return the Blackberry.
DJ Khaled Premieres Star-Studded Video for 'All I Do Is Win' RemixJune 28, 2010 04:06:17
Take a look at the Dream Team who are claimed to have contributed 'a significant level of greatness to the hip-hop culture' when they are trading verses with each other in this clip.
Jay-Z and Beyonce Knowles Lead 2010 BET Awards NominationsMay 19, 2010 09:05:32
Also scoring multiple nods are Alicia Keys, Trey Songz, Melanie Fiona, Drake, Nicki Minaj and Young Money.
Fabolous Describes Twitter Dispute With Jim Jones as JokeApril 03, 2010 02:04:06
Fabolous brushes off his Twitter war of words with Jim Jones following Jones' plagiarism accusation, saying 'But I had fun with it, I made some jokes.'


text:
Trillium pussilum
gerhard(Z6/7)December 1, 2013
Hi, is anybody growing any of the following trilliums?
Trillium pussilum var alabamicum, var. monticola, var. texanum or var. virginianum.
I have the Alabama one, a gift from a friend. They have bloomed sparsely but regularly since I received them three years ago. Earlier in the summer we had a great deal of heavy rain, so I am hoping they are still where I planted them, near a ditch that handles run-off and is usually placid but was very full this summer.


text:
Page 1
Excipients Differentially Influence the Conformational Stability
and Pretransition Dynamics of Two IgG1 Monoclonal Antibodies
1Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence,
Kansas 66047
2Formulation Sciences, MedImmune, Gaithersburg, Maryland 20878
Received 27 February 2012; revised 11 April 2012; accepted 20 April 2012
Published online 11 May 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23187
ABSTRACT: Since immunoglobulins are conformationally dynamic molecules in solution, we
studied the effect of stabilizing and destabilizing excipients on the conformational stability and
dynamics of two IgG1 monoclonal antibodies (mAbs; mAb-A and mAb-B) using a variety of
biophysical approaches. Even though the two mAbs are of the same IgG1 subtype, the unfolding
patterns, aggregation behavior, and pretransition dynamics of these two antibodies were strik-
ingly different in response to external perturbations such as pH, temperature, and presence of
excipients. Sucrose and arginine were identified as stabilizers and destabilizers, respectively, on
the basis of their influence on conformational stability for both the IgG1 mAbs. The two excipi-
ents, however, had distinct effective concentrations and different effects on the conformational
stability and pretransition dynamics of the two mAbs as measured by a combination of differen-
tial scanning calorimetry, high-resolution ultrasonic spectroscopy, and red-edge excitation shift
fluorescence studies. Stabilizing concentrations of sucrose were found to decrease the internal
motions of mAb-B, whereas arginine marginally increased its adiabatic compressibility in the
pretransition region. Both sucrose and arginine did not influence the pretransition dynamics
of mAb-A. The potential reasons for such differences in excipient effects between two IgG1
mAbs are discussed. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association
J Pharm Sci 101:3062–3077, 2012
proteins; immunoglobulins; stability; pretransition dynamics; protein aggrega-
tion; excipients; protein formulation; biophysical techniques
Proteins in solution are inherently conformationally
dynamic molecules composed of atoms that are in a
state of constant motion at ambient temperatures.1
At equilibrium, the native form of a protein is be-
lieved to sample a statistical ensemble of intercon-
verting microstates that undergo continuous fluctu-
ations, resulting in protein motions on the spatial
scale of sub-nanometer to tens of nanometers and
a temporal scale of femtoseconds to hours.2Pro-
tein dynamics are known to influence a wide variety
of biological processes including folding,1enzymatic
Additional Supporting Information may be found in the online
version of this article. Supporting Information
Corresponding to: C. Russell Middaugh (Telephone: +785-864-
5813; Fax: +785-864-5814; E-mail: email@example.com)
Journal of Pharmaceutical Sciences, Vol. 101, 3062–3077 (2012)
© 2012 Wiley Periodicals, Inc. and the American Pharmacists Association
activity,3,4signaling,5allostery,6ligand binding,7–9
and stability.10Several studies suggest that exam-
ining the dynamics of proteins could play a role in
elucidating more complex correlations that may exist
between protein stability and function.11–15
Changes in solution properties (e.g., pH, tem-
perature, ionic strength, and the presence of co-
solvents) as well as the structure of water itself
(predominantly in the hydration layer) may signif-
icantly influence the structure, stability, dynamics,
and function of biologically and pharmaceutically
important proteins.16–21A variety of biophysical
techniques such as X-ray crystallography,22,23nu-
clear magnetic resonance,3,24–26neutron scattering,23
isotope exchange,27ultrasonic spectroscopy,10,20,28–31
and pressure perturbation calorimetry20,32–35have
been employed to probe fluctuations in the inter-
nal motions of proteins and/or their surrounding sol-
vent. Numerous lower resolution techniques such as
Page 2
ultraviolet (UV)-absorption, fluorescence, circular
dichroism (CD), and light scattering among oth-
ers have commonly been employed to characterize
higher order structures, hydrodynamic properties,
and conformational stability of proteins.10,36–44Data
from these multiple biophysical techniques can be
combined in a vector-based stress/response method
known as an empirical phase diagram45–47(EPD). An
EPD displays distinct colored regions, which repre-
sent different conformational states of proteins and
other macromolecular systems as a function of so-
lution conditions such as pH and temperature. In
a recent study,47an EPD was generated for an
IgG1 monoclonal antibody (mAb-B) based on tech-
niques sensitive to the dynamic properties of pro-
teins such as high-resolution ultrasonic spectroscopy
(HR-US), pressure perturbation calorimetry, red-edge
excitation shifts (REES), and time-resolved fluores-
cence spectroscopy. The results showed a more com-
plex pattern of apparent structural transitions at
lower temperatures in the pretransition region (be-
low any detectable unfolding event) compared with
an EPD generated from biophysical data using static
(time-averaged) measurements such as CD, steady-
state fluorescence spectroscopy, and light scattering.
The pretransition region is defined as a temperature
range over which the change in parameters tradition-
ally used to evaluate a protein's secondary structure,
tertiary structure, and conformational stability does
not deviate from a continuous change with tempera-
ture, as studied by methods such as CD, fluorescence
spectroscopy, and differential scanning calorimetry
(DSC). A better understanding of any relationship
between conformational stability and dynamics, espe-
cially in the pretransition region, may be important
to our understanding of the development and formu-
lation of biopharmaceutical drugs such as monoclonal
antibodies (mAbs).
Monoclonal antibodies are an important class of
dynamic, Y-shaped proteins that are good models
for studying the interrelationships between confor-
mational stability and dynamics. The two Fab do-
mains of immunoglobulins are connected to the Fc
domain by a highly flexible proline-rich hinge region,
which is believed to affect the structure and dynam-
ics of immunoglobulins.48,49Various analytical tech-
niques have been used to study the flexibility and
dynamics of antibodies.20,27,47–54Different molecules
within an immunoglobulin subclass, despite their
overall similarity in structure and sequence homol-
ogy, may display significant differences in their con-
formational stability, flexibility, and dynamics. The
conformational stability of antibody drugs, formu-
lated at both low and high concentrations, is signifi-
cantly influenced by environmental and formulation
factors during manufacturing, long-term storage, and
administration.55–57The effect of these factors on pro-
tein dynamics, however, has not been examined to
any great extent. It is therefore important to not only
better understand any relationship between confor-
mational stability and dynamics for different mAbs,
but to also examine the effect of various environmen-
tal factors (e.g., pH, temperature, excipients, etc.) on
their conformational stability and dynamics.
In this study, the effect of stabilizing and destabi-
lizing excipients on conformational stability and in-
tramolecular protein dynamics of two different IgG1
mAbs (mAb-A and mAb-B) is compared to further
understand the relationships between stability and
The IgG1 mAbs (mAb-A and mAb-B) were provided
by MedImmune (Gaithersburg, Maryland). The stock
protein solutions were stored as received at 2◦C–8◦C.
The dialysis of stock protein solutions was carried out
overnight (at 4◦C) using a 10kDa molecular weight
cutoff dialysis cassette (Pierce, Rockford, Illinois) into
20mM citrate–phosphate buffer at pH values rang-
ing from 3 to 8 at one unit intervals, unless otherwise
noted. The final ionic strength of the buffer was ad-
justed to 0.1 using NaCl. All of the buffer components
and other chemicals were purchased from Sigma
(St. Louis, Missouri) and Fisher Scientific (Pitts-
burgh, Pennsylvania). The protein concentration was
measured at room temperature by absorbance mea-
surement at 280nm using an extinction coefficient
1.45mL/(mg cm) in an Agilent 8453 UV–visible
spectrophotometer (Palo Alto, California), and di-
luted to the final concentration as indicated in each
Steady-State Intrinsic (tryptophan) and Extrinsic
(1-anilino naphthalene-8-sulphonate) Fluorescence
Spectroscopy, Far-UV CD, and OD350nmTurbidity
For each of these four techniques, the experimen-
tal method as applied to mAbs has been described
High-Resolution Ultrasonic Spectroscopy
Ultrasonic measurements20,47,58–60were performed
using an HR-US 102 Spectrometer (Ultrasonic Sci-
entific, Dublin, Ireland) with a frequency range of
2–18MHz and a resolution of 0.2mm/s for velocity
and 0.2% for attenuation measurements. The sam-
ple and reference cells contained 1mL of protein and
corresponding buffer solution, respectively. The dif-
ferential velocity and attenuation were monitored at
12MHz from 10◦C to 85◦C and pH 3–8 using 5mg/mL
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 9, SEPTEMBER 2012
Page 3
of mAb-A. The temperature of the cells was con-
trolled by a Phoenix P2 water circulator (Thermo
reference solutions were thoroughly degassed before
each measurement. Appropriate amounts of sucrose
or arginine were added to both protein and buffer so-
lutions while evaluating excipient effects. Data were
analyzed using HRUS v18.104.22.168 software (Ultra-
sonic Scientific, Dublin, Ireland). The coefficient of
adiabatic compressibility (βs) was determined using
the following equations20:
βs= −1
?(β/β0) − V0
s = −
V0=ρ − c
; and v0= lim
β and β0are the adiabatic compressibility of the solu-
tion and buffer, respectively; ρ and ρ0are the density
of the solution and the corresponding buffer; ν0is the
partial specific volume of the IgG; V0is apparent vol-
ume fraction of the buffer; and c is the protein concen-
tration. The adiabatic compressibility of the sample
velocity (μ) by the Laplace equation,61β = 1/ρμ2. The
effect of excipients on the compressibility of mAb-A
and mAb-B was studied similarly using solution con-
ditions described later in the text.
The density of protein samples (5mg/mL) and cor-
responding buffer solutions was measured using a
DMA-5000 high-precision densitometer (Anton Paar,
Graz, Austria) at a precision of 1× 10−6g/cm3and
0.001◦C. The densities of degassed solutions were
measured from 5◦C to 55◦C at 2.5◦C intervals. The
instrument was calibrated daily with dry air and de-
gassed water before analysis. For the excipient stud-
ies, both the protein sample and corresponding buffer
solution contained equal predetermined quantities of
each excipient.
Differential Scanning Calorimetry
The differential scanning calorimetric studies were
performed using a MicroCal VP-Capillary DSC
with an autosampler (MicroCal, Northampton, Mas-
sachusetts). The pH (pH 3–8 at unit intervals) ex-
periments for mAb-A and mAb-B were performed us-
ing 1mg/mL of protein in 20mM citrate–phosphate
buffer (I = 0.1 adjusted by the appropriate addition
of NaCl). The temperature ramp was programmed
from 10◦C to 90◦C at a scanning rate of 60◦C/h and a
filtering period of 16s. Protein thermograms were ob-
tained by subtracting the corresponding buffer blank
from the sample thermogram. The transition mid-
points were obtained by determining the baseline us-
ing linear or cubic functions, normalizing it to protein
concentration, and fitting the processed thermogram
to a non-two-state unfolding model. The endothermic
peak maximum of the heat capacity was considered
to be the apparent transition midpoint (TM) for the
individual peaks that could be deconvoluted from the
thermogram. The effect of varying concentrations of
excipients was studied similarly at pH 4 and pH 4.5
for mAb-A and mAb-B, respectively.
Empirical Phase Diagrams
Empirical phase diagrams are constructed to visu-
ally represent changes in the structural45,47and
dynamic47properties of proteins in the form of colored
diagrams as a function of solution variables such as
pH and temperature. The rationale and methodology
separate EPDs were constructed using mAb-A. For
the first EPD, experimental data as a function of pH
and temperature from the following static biophysical
techniques were used: intrinsic tryptophan (Trp) flu-
orescence intensity, Trp peak position shifts, static
light scattering intensity, 1-anilino naphthalene-8-
sulphonate (ANS) fluorescence intensity, CD at 217
nm, and optical density at 350nm (OD350nm) values.
The second EPD was constructed to include mea-
surements of dynamic properties of mAb-A by adding
compressibility data from HR-US. The latter reflects
changes in global dynamics as a function of pH and
temperature. These two EPDs for mAb-A are com-
pared with previously published results for mAb-B.47
Different protein concentrations were used for dif-
ferent techniques (e.g., 0.1mg/mL for fluorescence
measurements and 5mg/mL for ultrasonic measure-
ments), and the concentration was chosen to obtain
higher signal-to-noise in individual measurements.
dependent) experiments, the differences in protein
concentration do not alter the pretransition region
significantly and hence should not influence the con-
formational stability and dynamics of the proteins as
measured by these techniques.
Excipient Screening
Intrinsic Trp fluorescence and static light scatter-
ing were used to screen a generally-regarded-as-safe
(GRAS) library of excipients to identify compounds
that either increased or decreased the stability of
mAb-B, as determined by changes in the TMof mAb-B
unfolding. The concentrations of excipients used were
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 9, SEPTEMBER 2012DOI 10.1002/jps
Page 4
higher than those commonly used in protein formu-
lations to facilitate screening of excipients. The TM
was determined from a sigmoidal fit of the first tran-
sition in intrinsic fluorescence intensity versus tem-
perature plots. The data were acquired between 10◦C
and 90◦C in increments of 2.5◦C. Static light scatter-
ing was used to assess the propensity of the IgG1 an-
tibody to aggregate. Prior to experimentation, mAb-
B was dialyzed into 20mM citrate–phosphate buffer
(containing NaCl to adjust I = 0.1) at pH 4.5. These
stress conditions were selected on the basis of the re-
duced stability of mAb-B at this pH,47which should
facilitate the identification of stabilizing compounds.
The protein concentration employed was 0.1mg/mL.
A few selected candidate stabilizers and destabilizers
from this primary screen were used to further evalu-
ate their effect on the second IgG1 molecule (mAb-A),
either by the fluorescence method and/or DSC.
Red-Edge Excitation Spectroscopy
Red-edge excitation is a characteristic property of po-
lar fluorophores, which exhibit excitation wavelength
dependent emission spectra.62,63This phenomenon
depends upon motional restriction of the environ-
ment of fluorophores. The steady-state fluorescence
measurements of REES were performed using a PTI
Quanta Master Spectrophotometer (Photon Technol-
ogy International, Inc., Lawrenceville, New Jersey).
The excitation and emission slit widths were set
to 2.5 and 3nm, respectively. The emission spectra
(300–400nm) were collected using different excita-
tion wavelengths from 292 to 308nm at 4nm inter-
ied in the absence and presence of selected excipients
using a 1cm pathlength quartz cuvette. A temper-
ature ramp from 10◦C to 70◦C at 2.5◦C increments
was used with an equilibration time of 3min. An ap-
propriate blank spectrum was subtracted from the
sample spectrum. The emission peak position (and in-
tensity) was determined by a mean spectral center of
mass (MSM) method, which increased reproducibil-
ity and signal-to-noise ratio and thus improved our
ability to measure the relative shifts in peak posi-
tion in the presence of excipients. The peak position
maxima obtained by the MSM method is approxi-
mately 8–10nm higher than the actual peak position
obtained by derivative analysis.
Characterization of Higher Order Structure,
Conformational Stability, and Dynamics of mAb-A as a
Function of pH and Temperature
Static (Time-Averaged) Measurements
tion measurements of mAb-A as a function of pH and
temperature are shown in Figures 1a–1f and Supple-
mentary Figures S1a–S1f. A well-defined structural
transition occurs in mAb-A at 65◦C–75◦C over the
pH range of 5–8 as detected by increases in Trp flu-
orescence intensity (Fig. 1a) and a red shift of emis-
sion peak maximum (Fig. 1b). The onset temperature
(Tonset) of unfolding at pH 5 starts approximately at
60◦C with a broad unfolding curve (Fig. 1b). The early
Tonsetat pH 5 is accompanied by an increase in ANS
fluorescence intensity (Fig. 1c), suggesting the expo-
sure of apolar sites. The presence of intermolecular
β-structure-rich structures at pH 5–8 is also detected
at approximately 63◦C–78◦C as suggested by a de-
crease in the CD signal (Fig. 1f). The unfolding event
at pH 5–8 leads to further aggregation, which is ap-
parent from an increase in light scattering intensity
(Fig. 1d) and OD350nm (Fig. 1e) measurements. The
of pH 5–8, however, follows an opposite trend com-
pared with the Tonsetof aggregate formation (Figs. 1d
and 1e). For example, the Tonsetof unfolding is low-
est (∼60◦C) at pH 5 (followed by pH 6< pH 7< pH
8), whereas the Tonset of aggregation is the highest
(∼85◦C) at pH 5 (followed by pH 6> pH 7> pH 8). In
addition, at pH 4, the overall structural changes ob-
served for mAb-A as a function of temperature were
similar (Figs. 1b, 1c, and 1f) to that at pH 5 up to ap-
proximately 70◦C, although the transitions were ob-
served at lower temperatures. The CD signal at pH 4,
however, continues to decrease above 75◦C, suggest-
ing that the intermolecular interactions continue to
increase with increases in temperature. The behavior
other pH values below 65◦C, where the protein man-
ifests multiple distinct structural transitions appar-
ent from ANS fluorescence intensity change (Fig. 1c),
Trp peak position shifts (Fig. 1), and CD (Fig. 1f).
No increase in light scattering intensity (Fig. 1d) or
OD (Fig. 1e) was observed at pH 3 and 4 up to 90◦C.
Fluorescence emission spectra (intrinsic and ANS) for
mAb-A at 15◦C, 35◦C, and 60◦C are shown as a func-
tion of pH in Supplementary Figures S1a–S1f. These
data suggest that mAb-A retains its native-like struc-
ture (Supplementary Figs. S1a, S1b, S1d, and S1e)
at temperatures in the pretransition region (15◦C
and 35◦C) compared with results at higher temper-
atures (e.g., 60◦C) that results in structural disrup-
tions (Supplementary Figs. S1c and S1f). Upon exci-
tation at 295nm, the intrinsic Trp fluorescence emis-
sion represents the average emission signal from all
the Trp residues (∼22) present in the IgG1 molecules
used in this study.
High-Resolution Ultrasonic Spectroscopy
The global dynamics of mAb-A were studied by de-
termining compressibility (volume fluctuations with
changes in pressure) as a function of pH and
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Page 5
Figure 1.
antibody (mAb-A) as measured by a variety of biophysical techniques: (a) intrinsic tryptophan
fluorescence intensity, (b) intrinsic tryptophan fluorescence peak position shifts, (c) ANS fluo-
rescence intensity, (d) static light scattering, (e) OD350nm, and (f) CD signal at 217nm.
Effect of pH and temperature on conformational stability of an IgG1 monoclonal
pressibility of mAb-A was calculated as a function
of pH and temperature by determining the relative
changes in the ultrasonic velocity between the sam-
ple and reference. The adiabatic compressibility of
mAb-A was found to increase as a function of temper-
ature at all pH values. Plots of adiabatic compressibil-
ity versus temperature, however, show a unique non-
linear increase in the pretransition regions (<45◦C)
starting at pH 4 and above. These HR-US deviations
occur at lower temperatures than the respective Tonset
and TM1 values as measured by DSC (see next sec-
tion). This nonlinear dependence of adiabatic com-
pressibility may therefore reflect some form of change
in the global dynamics of mAb-A in the pretransition
EPDs for mAb-A
The EPD constructed for mAb-A using the time-
averaged measurements is presented in Figure 3a.
A broad structural transition is apparent between
60◦C and 75◦C for pH 5–8. At pH 4, two distinct
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 9, SEPTEMBER 2012DOI 10.1002/jps
Page 6
Figure 2.
sured by HR-US. The straight lines along the data points are a visual aid for comparison of
pretransition regions. The Tonsetand TMvalues represent the initiation and peak maximum for
the first thermal transition as determined separately by DSC.
Adiabatic compressibility of mAb-A as a function of pH and temperature as mea-
structural transitions occur at approximately 50◦C
and approximately 70◦C. The contributions from ANS
fluorescence intensity changes (Fig. 1c) and CD sig-
nals (Fig. 1f) may contribute the most to these appar-
ent transitions at pH 4. The transitions in mAb-A at
pH 3 start at approximately 20◦C with multiple sub-
sequent minor transitions observed every 10◦C–15◦C.
The EPD in Figure 3a has been divided into three
distinct phases, i.e., Phases I, II, and III, represent-
ingregions ofstable,unstable,andaggregated formof
(mAb-B) was able to detect an additional transition
region in the low-temperature (<45◦C) region aris-
ing from contributions of the adiabatic compressibil-
ity measurements. An EPD using mAb-A was there-
fore constructed combining static and compressibility
measurements as a function of pH and temperature
(Fig. 3b). REES data were not included in the EPD
because of lower resolution of these data at higher
temperatures. This "dynamic" EPD for mAb-A shows
an additional transition region (Phase 1?) at pH ≥
4 and at temperatures below 45◦C compared to the
static EPD (Fig. 3a). This EPD with mAb-A, together
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Page 7
Figure 3.
static (time-averaged) biophysical techniques alone, and (b) data from static techniques in (a)
in conjunction with HR-US data. Static biophysical techniques data from intrinsic tryptophan
fluorescence intensity and peak position shifts, static light scattering intensity, extrinsic ANS
fluorescence intensity, circular dichroism at 217nm, and OD350nmvalues were used. A continu-
ous color in the phase diagram represents a single structural state of the protein. Transitions
in the proteins' structure are manifested by changes in color.
Empirical phase diagrams for mAb-A as a function of pH and temperature using (a)
with the previously published47results with mAb-B,
suggests that ultrasonic measurements provide addi-
tional information about conformational fluctuations
and flexibility in IgG1 that are not apparent with the
use of conventional biophysical techniques alone, es-
pecially in the pretransition regions. The presence of
additional structural effects in the pretransition re-
gions of the two IgG1 antibodies at approximately pH
5–8 emphasizes the need for developing a better un-
derstanding of the effect of formulation components
not only on equilibrium conformational stability, but
also on the dynamic properties of protein therapeutic
drugs in solution.
Thermal Stability (TM) of mAb-A and mAb-B
Differential scanning calorimetry is routinely used
to study the thermal stability of antibodies20,64by
measuring the differential heat capacity to determine
midpoints of thermal unfolding events (TMs). DSC
was used to directly compare the conformational sta-
bility of mAb-A and mAb-B as function of solution pH
(Fig. 4; Supplementary Table 1). Three distinct con-
formational transitions (TM1, TM2, and TM3) were
apparent for mAb-A at pH 3–7, albeit at variable
temperatures. Only two major structural transitions,
however, were readily observable at pH 8 (Fig. 4b).
In contrast, mAb-B showed only two distinct transi-
tions in the pH range of 5–8 by DSC (TM2 and TM3
in Figs. 4c and 4d). In the case of mAb-B at pH 3 and
4, an additional lower temperature transition (TM1)
was detected.
Screening of a GRAS Library of Excipients
To better understand the effect of formulation ex-
cipients on conformational stability and global dy-
namics of these two IgG1 mAbs, a first set of ex-
periments screened a GRAS library of excipients to
identify potential stabilizing and destabilizing excip-
ients using mAb-B. The EPDs for mAb-A (Fig. 3) and
mAb-B (Ramsey et al.,47Fig. 3) indicate conforma-
tional instability in the range of 55◦C–65◦C at pH
approximately 4–4.5. The intrinsic Trp fluorescence
intensity method was utilized to identify stabilizing
excipients (?TM) under these accelerated pH condi-
tions that altered the stability of mAb-B's tertiary
structure (Table 1). This methodology allowed for
determination of the protein unfolding temperature
(TM) as well as an assessment of aggregation behav-
ior (Table 1). Sugars and polyols in general increased
the TMof mAb-B, whereas amino acids such as argi-
nine and histidine lowered the transition tempera-
ture. A few selected candidate stabilizers (sucrose,
dextrose, and mannitol) and destabilizers (arginine)
were then tested with mAb-A (using DSC) at pH 4
to identify common excipients that would either sta-
bilize or destabilize both of the IgG1 antibodies (data
not shown). On the basis of their effects on the TMval-
ues, as measured by fluorescence spectroscopy with
mAb-B and DSC with mAb-A, sucrose was selected as
a representative candidate stabilizer, whereas argi-
nine was used as a destabilizing excipient for both
proteins. All subsequent studies evaluating the effect
of excipients on conformational stability and pretran-
sition dynamics were performed at pH 4 and pH 4.5
for mAb-A and mAb-B, respectively. These solution
conditions were selected because the magnitude of
stabilizing and destabilizing effects of these two ex-
cipients under neutral pH conditions were smaller
than the chosen more acidic pH solution conditions.
The selection of lower pH, however, does not preclude
our ability to study the effect of excipients on the
pretransition conformational dynamics of the mAbs
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 9, SEPTEMBER 2012DOI 10.1002/jps
Page 8
Figure 4.
unfolding values (TM1, TM2, TM3) for mAb-A (b) and mAb-B (d) as a function of pH. Error bars
cannot be seen in (b) and (d) because they are within the symbols. See Supplementary Table 1
for summary of TMresults and their corresponding SD values.
Representative differential scanning calorimetric thermograms for mAb-A (a) and
because the Tonsetof unfolding for both the proteins
under these conditions is still ≥ 45◦C.
Effect of Arginine and Sucrose on Conformational
Stability and Dynamics of IgG1 mAb-A and mAb-B
Effect on Conformational Stability
The effect of sucrose and arginine on the confor-
mational stability of mAb-A and mAb-B was stud-
ied using DSC and intrinsic Trp fluorescence spec-
troscopy (Figs. 5 and 6; Supplementary Table 2). DSC
and fluorescence measurements were used to deter-
mine the effect of excipients on the overall thermal
stability and tertiary structure stability, respectively.
Fig. 5a shows a representative DSC thermogram
for mAb-B at pH 4.5. Three distinct transitions
were detected for mAb-B at pH 4.5 with TM1 being
the first low-temperature transition, TM2 the sec-
ond transition, and TM3 the third transition seen
at the highest temperature. Similarly, three distinct
transitions were observed for mAb-A at pH 4 (data
not shown). Arginine (up to ∼300mM) was found
to destabilize both mAb-A (Fig. 5c; Supplementary
Table 2a) and mAb-B (Fig. 5e; Supplementary
Table 2c) in a concentration-dependent manner. Su-
crose (up to ∼500mM) showed a concentration-
tary Table 2d).
One goal of a protein formulation strategy would
be to identify excipients that stabilize the first con-
formational transition (which is typically the unfold-
ing of CH2 domain for IgG1) and inhibit subsequent
protein unfolding. Figure 5b shows the effect of argi-
nine and sucrose concentration on the TM1 values
for both mAb-A and mAb-B. Arginine was more po-
tent at destabilizing mAb-A than mAb-B. For exam-
ple, to achieve approximately 2.5◦C destabilization,
a lesser amount of arginine (highlighted with a rect-
angle) was required for mAb-A compared to mAb-B.
Sucrose, however, was a more potent stabilizer for
mAb-B compared to mAb-A. As shown in Figure 5b,
to achieve approximately 2.5◦C stabilization, a lesser
amount of sucrose (highlighted with a rectangle) was
required for mAb-B compared to mAb-A.
Figure 6 shows the effect of the two excipients on
the tertiary structure stability of mAb-A (Fig. 6a) and
mAb-B (Fig. 6b) as monitored by Trp peak position
shifts as a function of temperature. Arginine does
not influence the tertiary structure stability of mAb-
A throughout the temperature range examined. For
mAb-A in presence of sucrose, however, the protein
showed blue-shifted Trp peak positions above 25◦C,
suggesting that aromatic residues are shielded from
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Page 9
Table 1.
Spectroscopy Using 0.1mg/mL Protein at pH 4.5 in 20mM Citrate–Phosphate Buffer (Containing NaCl, I = 0.1 and Indicated Level of
Effect of Excipients on Thermal Unfolding Temperature (TM) of mAb-B as Monitored by Intrinsic Tryptophan Fluorescence
Aggregation Inhibition (%)c
IgG1 (mAb-B)
Malic acid
Aspartic acid
Lactic acid
Pluronic F-68
Sodium citrate
Brij 35
Tween 20
Tween 80
Dextran T40
Glutamic acid
Neutral excipients
?TMand aggregation inhibition (%) columns represent the change in midpoint of thermal unfolding and change in aggregation, respectively, for mAb-B in
presence of excipients.
aExcipient concentrations are higher than those commonly used in formulations to facilitate excipient screening.
bThe TMmeasurements were made within the standard deviation of ±0.5◦C.
cThe aggregation inhibition (%) represents a mean of three measurements with a standard deviation of ±2.5% for excipients that inhibited mAb-B
aggregation. NC is a group of excipients that resulted in no change (±10%) in the aggregation behavior of mAb-B. ND is a group of excipients that increased
mAb-B aggregation.
the solvent. In the case of mAb-B, in the absence of
excipients, the protein shows a red shift in Trp peak
position (Tonset∼45◦C) upon thermal unfolding, indi-
cating exposure of aromatic residues to the solvent
(Fig. 6b) with increasing temperature. Sucrose was
found to stabilize, whereas arginine destabilized the
tertiary structure of mAb-B in terms of both Tonset
and TM.
Effect on Protein Dynamics
The effect of sucrose and arginine on the global dy-
namics of mAb-A and mAb-B was first examined by
US as shown in Figure 7. The measurements of pro-
tein compressibility are directly related to the fluctu-
ations in volume of the protein, thereby reflecting a
7a shows that the adiabatic compressibility increases
with temperature for both mAb-A (pH 4) and mAb-
B (pH 4.5). An increase in compressibility suggests
that the relative difference in the ultrasonic velocity
between the sample and the reference is decreased,
whereas the absolute value of the velocity increases
as a function of temperature. It is well known that
mobile or less structured molecules possess a lower
elastic modulus compared to rigid, more structured
species. This results in a decrease in sound veloc-
ity through the unstructured or mobile material. A
higher compressibility value is therefore indicative
of a less structured and/or a more dynamic protein.
As seen in Figure 7a, the compressibility of mAb-A
is relatively greater than mAb-B in the temperature
range 10◦C–50◦C, that is, before any major detectable
conformational transitions.
The effect of sucrose and arginine on mAb-A com-
pressibility as a function of temperature is shown in
Figure 7b. Sucrose does not significantly influence the
compressibility of mAb-A in the pretransition region,
whereas arginine marginally decreases the compress-
ibility of mAb-A, especially in the pretransition re-
gion. Figure 7c represents the effect of sucrose and
arginine on the compressibility of mAb-B. The com-
pressibility of mAb-B in the presence of arginine was
found to be marginally increased in the pretransition
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 9, SEPTEMBER 2012 DOI 10.1002/jps
Page 10
Figure 5.
measured by DSC: (a) representative DSC thermograms for mAb-B (pH 4.5) with TM1, TM2,
and TM3; (b) effect of different concentrations of excipients on TM1 for mAb-A and mAb-B. The
box represents the effective concentration of excipient required to have approximately 2.5◦C of
effect. Plots of TMvalues for mAb-A (c and d) and mAb-B (e and f) in the presence of varying
concentrations of arginine (c and e) and sucrose (d and f). Error bars often cannot be seen
because they fall within the dimensions of the data points.
Effect of sucrose and arginine on conformation stability of mAb-A and mAb-B as
region. The significant increase in the compressibility
of mAb-B in the presence of arginine above 55◦C can
be most likely be explained by the formation of highly
compressible, irreversible aggregates. The compress-
ibility of mAb-B in the presence of sucrose was sig-
nificantly reduced throughout the range of temper-
atures used in this study, but predominantly in the
pretransition region. The lowering of compressibil-
ity suggests that the global dynamics of mAb-B are
dampened in presence of stabilizing concentrations of
Figure 8 shows the REES results for mAb-A and
mAb-B in the presence of sucrose and arginine.
The REES is a phenomenon in which there is a
shift in emission spectra maxima upon red-edge
excitation.62,63Such an effect is primarily seen when
the lifetime of solvent relaxation is equal to or larger
than the lifetime of the fluorophore of interest. The
solvent reorientation or relaxation around an excited
state fluorophore is influenced by dynamic motions
within proteins and solvent fluctuations around the
fluorophore's environment. At 10◦C, both mAb-A and
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Page 11
Figure 6.
sition shifts for (a) mAb-A and (b) mAb-B as a function of
temperature using fluorescence spectroscopy.
Effect of sucrose and arginine on Trp peak po-
mAb-B show red-edge shifts in the absence of excipi-
ents, suggesting that the fluorophores examined (Trp
in this case) are in an environment wherein the life-
time of solvent relaxation is either equal to or longer
than the lifetime of the fluorophore. Such a system is
suitable for studying the effects of excipients on the
conformational flexibility and dynamics of proteins by
monitoring their effect on the magnitude of any ob-
served red-edge shifts. Since the REES effect occurs
because the longer wavelength excitation results in
photoselection of fluorophores that are strongly inter-
acting with polar solvent molecules in their vicinity, a
less dynamic (or more rigid) fluorophore environment
will lead to a decrease in solvent relaxation of the flu-
orophore and thus the magnitude of red-edge shifts
will increase. As shown in Figures 8a and 8b, neither
sucrose nor arginine altered the red-edge shifts ob-
served in mAb-A in the temperature range spanning
the pretransition region of the antibody. This result
suggests that these two excipients do not significantly
affect the global dynamics of mAb-A in which the local
environment around Trp residues was sampled. By
contrast, concentrations of sucrose that stabilized the
Figure 7.
pressibility of mAb-A and mAb-B as measured by HR-US:
(a) comparison of compressibility of mAb-A and mAb-B in
the pretransition region, (b) effect of arginine and sucrose
on adiabatic compressibility of mAb-A, and (c) effect of argi-
nine and sucrose on adiabatic compressibility of mAb-B.
Effect of sucrose and arginine on adiabatic com-
tertiary structure of mAb-B were found to increase
the magnitude of red-edge shifts in the pretransition
region (Fig. 8d). Arginine did not alter the magni-
tude of the red-edge shifts in the pretransition region
(<55◦C) of mAb-B (Fig. 8c).
Comparison of Higher Order Structure, Thermal Stability
Behavior, and EPDs Between mAb-A and mAb-B47
The biophysical data suggest that mAb-A undergoes
multistep unfolding upon thermal unfolding with
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 9, SEPTEMBER 2012DOI 10.1002/jps
Page 12
Figure 8.
b) and mAb-B (c and d) in the presence and absence of sucrose (b and d) and arginine (a and c).
Red-edge excitation shifts (REES) fluorescence measurements with mAb-A (a and
oligomeric structures at pH 4–8 at temperatures prior
to major unfolding/aggregation events. These inter-
molecular interactions appear to be accompanied
by shielding of aromatic residues from the solvent,
as suggested by the blue shift in Trp peak position
observed at pH 5–8 between approximately 45◦C and
65◦C (Fig. 1b). The Tonsetof unfolding for mAb-A was
inversely related to the Tonsetof aggregation between
pH 5 and 8. For example, mAb-A at pH 5 had the
lowest Tonset of unfolding but the highest Tonset of
aggregation as measured by ANS fluorescence inten-
sity and static light scattering (or OD), respectively
(Figs. 1c, 1d, and 1e). An initial increase in ANS
intensity (Fig. 1c) at pH 5 reaches a plateau above
65◦C. This result along with a continuous decrease in
the CD signal (Fig. 1f) at pH 5 above 65◦C suggests
that no additional apolar residues are exposed in
this temperature range. There is, however, an in-
crease in β-structure-rich intermolecular oligomeric
structures. These results suggest that mAb-A at pH
5 forms partially altered structures that are either
stable in solution and/or form oligomeric species that
are resistant to the formation of larger aggregates
that can be detected by static light scattering and
OD measurements. Furthermore, a steep increase
in ANS fluorescence intensity and increase in CD
signal (Figs. 1c and 1f) at pH 6–8 above 78◦C suggest
that additional aromatic residues are being exposed
and that the intermolecular β-structures begin to
dissociate. The Tonset for such a dissociation event
above 78◦C is found to be in the following order:
pH 8< pH 7< pH 6 (Fig. 1f). Once these structures
dissociate and additional aromatic residues are
exposed, the antibody may become more prone to
formation of irreversible aggregates. The detection of
larger aggregated species (Figs. 1d and 1e) follows
a similar trend as mentioned above (i.e., Tonset for
aggregation pH 8< pH 7< pH 6), arguing that a
dissociation of relatively stable oligomeric species
may precede the formation of larger aggregates in
solution at higher pH. The sudden drop in light
scattering intensity and OD (after an initial increase
at temperatures above 83◦C) indicates that the ag-
gregates eventually fall out of solution. At pH 3 and
4, mAb-A manifests blue-shifted Trp peak positions,
an increase in ANS intensity, and decreases in CD
signal which indicate conformational alterations at
much lower temperatures compared to higher pH
conditions. Nevertheless, the protein remains aggre-
gation resistant under these conditions. In contrast,
a single cooperative transition was observed in the
case of mAb-B47between 60◦C and 70◦C at pH values
ranging from 5 to 8 based on static measurements
(Fig. 1).47The unfolding event in mAb-B leads to
the exposure of aromatic residues and the protein
subsequently forms larger aggregates at pH 5–8
(Figs. 1e and 1f).47Furthermore, mAb-B at pH 5–8
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 9, SEPTEMBER 2012
Page 13
aggregates over a very narrow temperature range
as detected by static light scattering. The Tonset of
unfolding had no correlation with the Tonsetof aggre-
gation in this pH range (Figs. 1e and 1f).47In this
same study, mAb-B at pH 4 showed a broad unfolding
transition and aggregated to a lesser extent than at
pH 5–8, whereas the protein at pH 3 was found to
be resistant to formation of detectable aggregates. In
summary, these results from a variety of biophysical
measurements clearly show that IgG1 mAb-A and
mAb-B have distinct patterns of conformational
alterations and aggregation behavior in response to
changes in pH and temperature.
routinely display multiple conformational transitions
due to environmental stresses such as changes in pH
and temperature, which can be detected by DSC. A
number of previous DSC studies have assigned these
different transitions to the unfolding of antigen bind-
ing (Fab) region, crystallizable (Fc) region, or individ-
ual domains within Fab and Fc regions.65–67The pres-
ence of three transitions detected by DSC for mAb-A
(Figs. 4a and 4b) over a wide range of pH (3–7) cor-
related with spectroscopic measurements, which also
showed multiple transitions (Figs. 1b, 1c, and 1f). The
highest Tonsetof unfolding for mAb-A (Figs. 1b and 1c)
and the lowest Tonsetof aggregation (Figs. 1d and 1e)
at pH 8 indicate that the protein aggregates rapidly
upon unfolding. Such a concerted phenomenon may
explain only two major transitions that are apparent
at pH 8. In contrast, mAb-B was found to have two
major transitions at higher pH except at pH 3 and 4
(Figs. 4c and 4d). Our previous spectroscopic results47
using mAb-B have shown that it undergoes a single
cooperative unfolding transition at pH 5–8. There-
fore, the two main structural transitions (compared
with three transitions for mAb-A) detected by DSC for
mAb-B may suggest that the unfolding of one domain
leads to an immediate subsequent unfolding of other
domains within the mAb-B molecule. The additional
transitiondetected at pH3and 4byDSC (Fig.4d) was
also observed by other spectroscopic methods at sim-
ilar temperatures (Ramsey et al.,47Figs. 1b and 1f).
The thermal behavior and aggregation data for mAb-
B suggests that no detectable stable intermediates
are formed and that the protein undergoes concerted
thermal unfolding and subsequent aggregation under
these solution conditions.
In addition, comparison of structural features and
thermal stability profiles of the two mAbs can be com-
pared by their differences in the static and dynamic
EPDs, i.e., mAb-A (Fig. 3 in this work) and mAb-
B (Ramsey et al.,47Fig. 3). The static EPDs gener-
ated for mAb-A and mAb-B47were both able to detect
differences in conformational stability as a function
of temperature and pH. The dynamic EPD contain-
ing the additional compressibility results for mAb-A
(Fig. 3b) was able to detect changes in dynamic be-
havior in the pretransition region at a broader pH
range (pH 4–8) than mAb-B (Ramsey et al.,47Fig. 3),
wherein the dynamic EPD was found to contain addi-
tional regions in the pH range of 6–8. This difference
in the EPDs of mAb-A and mAb-B may potentially be
due to inherent differences between the two IgGs or
due to experimental differences such as: (1) the dy-
namic properties of mAb-B were studied above 20◦C
rather than 10◦C for mAb-A. Therefore, the confor-
mational fluctuations that may exist at lower tem-
peratures could be incompletely represented in the
EPD for mAb-B, and/or (2) ANS fluorescence results
for mAb-B (Ramsey et al.,47Fig. 1f) at lower pH val-
ues show a broad unfolding transition with a Tonsetof
approximately 37.5◦C. The smaller magnitude of dy-
namic fluctuations may therefore be obscured during
the mathematical data processing used to construct
the EPD.
Effects of Arginine and Sucrose on the Conformational
Stability and Pretransition Dynamics of mAb-A and
Arginine was found to be a destabilizer, whereas su-
crose was a stabilizer for both mAb-A and mAb-B in
a concentration-dependent manner, albeit at differ-
ent effective concentrations as determined by DSC
measurements. Arginine was a more potent destabi-
lizer for mAb-A compared to mAb-B, whereas sucrose
was more potent at stabilizing mAb-B than mAb-A
(Fig. 5b; Supplementary Table 2a). Arginine, how-
ever, did not perturb the tertiary structure stability
of mAb-A. Potential reasons for the destabilizing ef-
fect of arginine on mAb-A could either be the sup-
pression of intermolecular β-structure-rich oligomer
formation and/or promotion of its dissociation.68The
destabilization of such β-structure-rich intermediate
structures, which potentially stabilize partially al-
tered structures of mAb-A, may increase the propen-
sity of mAb-A to form larger aggregates. Further-
more, the blue shift observed in Trp peak position
as a function of temperature for mAb-A in the pres-
ence of sucrose suggests that this sugar may be stabi-
lizing the intermolecular β-structure-rich structures,
Both of these excipients, however, influenced the ter-
tiary structure stability of mAb-B. Thus, the effect
of arginine and sucrose on the global thermal stabil-
ity and tertiary structure suggest that both mAb-A
and mAb-B interact with the same excipients in a
different manner and at different effective concentra-
tions. This may in part be because of the inherent
differences in the physicochemical properties and the
unfolding processes between the two proteins, where
mAb-A appears to have a greater propensity to form
β-structure-rich structures thatstabilizethepartially
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 9, SEPTEMBER 2012DOI 10.1002/jps
Page 14
altered native structure before any global unfolding/
aggregation event. In contrast, mAb-B undergoes a
more cooperative unfolding process starting with dis-
ruption of its tertiary structure followed by an imme-
diate aggregation of the structurally altered protein.
It is generally accepted that experimentally deter-
mined adiabatic compressibility values are comprised
of positive contributions from the intrinsic compress-
ibility of a protein and a negative contribution from a
hydration component.61Depending upon the magni-
contribution, the apparent adiabatic compressibility
values may either be positive or negative. The lower
(negative at lower temperatures) adiabatic compress-
ibility values in the pretransition region for mAb-B
compared to mAb-A (Fig. 7a) suggest a combined ef-
fect of lower intrinsic compressibility and/or higher
hydration contribution to mAb-B. Such a combined
effect may result in stronger coupling of mAb-B con-
formational fluctuations to the fluctuations in the
surrounding hydration water and/or the proteins' en-
vironment compared to mAb-A. Sucrose did not af-
fect the compressibility of mAb-A in the pretransition
region. This result may be related to the lower po-
tency of sucrose as a stabilizer of mAb-A. Arginine
marginally lowered the compressibility of mAb-A re-
sulting in negative values in the pretransition region,
suggesting an increased contribution of hydration to
the apparent adiabatic compressibility values. In con-
compressibility values go from negative to positive at
lower temperatures, suggesting either an increase in
tion contribution. These effects, however, may not be
mutually exclusive. Arginine was found to destabilize
mAb-B by influencing its tertiary structure stability.
Such a destabilization effect may require perturba-
tion of the structure of water in the protein's hydra-
tion layer, potentially due to the chaotropic nature of
guanidinium group in arginine, resulting in a lower
hydration contribution to the apparent compressibil-
ity of mAb-B. This could explain the marginal in-
lower temperatures. Sucrose, however, significantly
decreased the compressibility of mAb-B especially in
the pretransition region. The negative values of mAb-
B compressibility in the presence of sucrose indicate
that the hydration component has a greater contribu-
the protein's surface may have a significant effect on
the dynamic behavior of mAb-B in the presence of su-
crose. Overall, these compressibility measurements
suggest that mAb-B exists in a less dynamic or a more
compact form due to the potent stabilizing effect of su-
crose, which may increase the ordering of water in the
hydration layer due to a preferential hydration mech-
anism. In summary, HR-US results show that effec-
tive concentrations of arginine and sucrose did not
significantly influence the dynamic behavior of mAb-
A in the pretransition region. Similarly, arginine did
not appreciably affect the pretransition dynamics of
mAb-B. Sucrose, however, significantly reduced the
dynamic behavior of mAb-B as indicated by the lower
compressibility values in the pretransition region.
Such a reduction in mAb-B pretransition dynamics
REES in the presence of stabilizing concentrations of
sucrose. The increase in magnitude of the red-edge ef-
fect suggests that solvent relaxation contributions in
the environment around the Trp residues in mAb-B
were reduced. This in turn suggests that the envi-
ronment around the Trp residues is rigidified in the
presence of sucrose, especially at lower temperatures,
potentially due to a reduction in the internal dynam-
ics of mAb-B. The inability of both arginine and su-
crose to affect the magnitude of red-edge effects for
mAb-A at different temperatures suggests that excip-
ients did not alter the dynamics of mAb-A in the im-
mediate environment of aromatic residues. Since the
conformational fluctuations in mAb-A and mAb-B are
differently coupled to solvent fluctuations, it is possi-
ble that excipients that modulate solvation properties
can influence the pretransition dynamics of proteins
whose conformational fluctuations are strongly cou-
pled to the surrounding solvent.
The two IgG1 mAbs used in this study exhibited
notable differences in their conformational stability
and dynamic properties as a function of pH, tem-
perature, and presence of excipients. Using a vari-
ety of biophysical techniques, these differences could
be summarized using an EPD approach. Sucrose and
arginine were found to influence the conformational
stability of mAb-A and mAb-B to varying extents at
different effective concentrations. These two excipi-
ents did not significantly influence the pretransition
REES studies. In contrast, for mAb-B, the effects of
stabilizing concentrations of sucrose on the compress-
ibility and the magnitude of red-edge effects suggest
that both the internal dynamics and the surrounding
solvent dynamics of mAb-B are influenced by sucrose
with this particular IgG1. These results show that
formulation components can have unique effects on
the dynamics of individual proteins within a single
IgG1 subclass, especially in the pretransition region.
Although the conformational stability and dynamics
results in this work highlight some of the differences
in physical behavior between two similar IgG mAbs,
themolecular originofthesedifferences remains tobe
DOI 10.1002/jpsJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 9, SEPTEMBER 2012
Page 15
elucidated. Such distinct effects of excipients on dy-
namics of therapeutic proteins may thus profoundly
influence the long-term storage stability and effi-
cacy of biopharmaceutical products and thus require
evaluation during preformulation and formulation
The authors would like to thank MedImmune for pro-
viding IgG1 molecules and financial support for this
work. Dr. Jae Hyun Kim is acknowledged for his
help in automating the data processing steps for
REES studies and for constructing EPDs. We thank
Dr. Hardeep Samra from Formulation Sciences De-
partment at MedImmune for helpful discussions and
critical review of this manuscript.
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text:
Homeland Security:
Transfer of Budgetary Resources to the Department of Homeland Security (DHS)
GAO-04-329R: Published: Apr 30, 2004. Publicly Released: May 4, 2004.
Additional Materials:
- Full Report:
- Accessible Text:
(202) 512-4636
Office of Public Affairs
(202) 512-4800
The enactment of the Homeland Security Act of 2002 led to the most substantial reorganization of the federal government since the 1940s by creating the Department of Homeland Security (DHS). Established in January 2003, DHS combined 29 program functions that were transferred from 10 agencies, referred to as the legacy agencies. In addition, legacy agencies transferred resources for support functions, such as offices of inspectors general and management and support. According to the President's proposal to create DHS, the reorganization was also designed to, among other things, achieve future savings through the elimination of redundancies among the transferred programs. Given the breadth and magnitude of the reorganization, questions have been raised among members of Congress and in the media about whether the creation of DHS would prompt increases in the size of the workforces at the agencies affected by the reorganization. This report responds to a request by the Chairman, House Committee on Transportation and Infrastructure, that we examine how the transfer of full-time equivalent (FTE) positions to DHS was conducted at the agencies involved in the transfer. Specifically, in this report, we (1) identify the budgetary resources and number of FTEs transferred from the legacy agencies to DHS, (2) compare the number of FTEs at DHS with the number of FTEs transferred from functions at legacy agencies, (3) identify differences between legacy agency fiscal year 2003 FTE balances after the transition and their fiscal year 2004 FTE requests, and (4) discuss any DHS efforts to identify opportunities to achieve personnel cost savings related to the reorganization.
The Office of Management and Budget (OMB) estimated that of the $38.28 billion transferred from 10 legacy agencies to DHS, $24.76 billion would support DHS's fiscal year 2003 needs, including funding for the 181,875 FTEs transferred from legacy agencies. Ninety-seven percent of the FTEs transferred were associated with seven program functions whose budget accounts were transferred to DHS in their entirety, including the functions of a former standalone agency, the Federal Emergency Management Agency. The nine remaining legacy agencies reported FTE decreases in their fiscal year 2003 budgets that generally matched OMB estimates of FTEs transferred to DHS. Only limited information was available to identify differences between legacy agency FTE balances after the transfer and their fiscal year 2004 requests. For the 24 budget accounts that were the source of the remaining 3 percent of FTEs transferred to DHS, the nine legacy agencies reported that, for their fiscal year 2004 requests: (1) 17 of 24 budget accounts included requests for FTEs less than or equal to the number of FTEs remaining after the transfer, and (2) 7 of 24 budget accounts included requests for FTEs greater than the number of FTEs remaining after the transfer. All legacy agencies reported that requested increases in FTEs were unrelated to the transfer of FTEs to DHS. DHS officials told us that while it was too early to identify personnel savings from the consolidation of legacy agency program functions in DHS, the department is beginning to examine some opportunities that may lead to such savings. They also said that these savings opportunities will have to be balanced with new departmental mission needs as yet to be determined.
Mar 31, 2015
Federal Protective Service:More Effective Management Needed in Delegating Security Authority to AgenciesGAO-15-271: Published: Mar 31, 2015. Publicly Released: Mar 31, 2015.
Mar 25, 2015
Aviation Security:TSA's Managed Inclusion Process Expands Passenger Expedited Screening, But TSA Has Not Tested Its Security EffectivenessGAO-15-465T: Published: Mar 25, 2015. Publicly Released: Mar 25, 2015.
Mar 24, 2015
Homeland Security:Action Needed to Better Assess Cost-Effectiveness of Security Enhancements at Federal FacilitiesGAO-15-444: Published: Mar 24, 2015. Publicly Released: Mar 24, 2015.
Mar 23, 2015
Border Security:Additional Efforts Needed to Address Persistent Challenges in Achieving Radio InteroperabilityGAO-15-201: Published: Mar 23, 2015. Publicly Released: Mar 23, 2015.
Mar 16, 2015
Homeland Security Acquisitions:DHS Should Better Define Oversight Roles and Improve Program Reporting to CongressGAO-15-292: Published: Mar 12, 2015. Publicly Released: Mar 16, 2015.
Mar 10, 2015
Government Publishing Office:Production of Secure Credentials for the Department of State and U.S. Customs and Border ProtectionGAO-15-326R: Published: Mar 10, 2015. Publicly Released: Mar 10, 2015.
Mar 6, 2015
Aviation Security:TSA Should Take Additional Action to Obtain Stakeholder Input when Modifying the Prohibited Items ListGAO-15-261: Published: Feb 4, 2015. Publicly Released: Mar 6, 2015.
Feb 26, 2015
Department of Homeland Security:Progress Made, but More Work Remains in Strengthening Management FunctionsGAO-15-388T: Published: Feb 26, 2015. Publicly Released: Feb 26, 2015.
Jan 27, 2015
Supply Chain Security:CBP Needs to Enhance Its Guidance and Oversight of High-Risk Maritime Cargo ShipmentsGAO-15-294: Published: Jan 27, 2015. Publicly Released: Jan 27, 2015.
Jan 12, 2015
Federal Facility Cybersecurity:DHS and GSA Should Address Cyber Risk to Building and Access Control SystemsGAO-15-6: Published: Dec 12, 2014. Publicly Released: Jan 12, 2015.
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text:
Can I "fool" my 11" induction hob to accept 8" pans?
I have decided to go with a shallower vent hood for aesthetic reasons in my small kitchen and plan to cook the "messies" (fry, long simmer, & boil) on the back induction hobs to compensate. Unfortunately, on my Bosch cooktop those back hobs are 5.75" and 11". All of my new larger induction pans/pots have 8" bottoms. These pans are the right size for my cooking needs so would prefer not to buy 11" if I can avoid it. An 8" pan on the large hob will heat up fine but has a constant anoying cyclic hum I can't live with. (The hum does not occur if the pans are sized to the correct burner.) Do you think placing a 18/8 SS waiter's tray from a restaurant supply store on the larger hob, then placing the 8" pan on top, would stop the cyclic hum? If so, would the pot still heat appropriately? The tray I'm considering is 13.75" round but has a slight raised edge so the flat bottom should be about 11". If this is a no go, does anyone have other suggestions of what I might try short of buying new pans? Thanks to anyone taking the time to read this and offer assistance.
The restaurant tray should heat up, certainly if it is responsive to a magnet. Heat will conduct into the pan on top. You might be able to fry bacon that way, but putting high power into the pan might prove difficult. And the pan may still vibrate.
There are relatively inexpensive induction capable cookwares that would probably be less trouble to acquire and use.
Boiling where there is minimal grease entrained in the water vapor could be done on a front hob and allowed to escape the hood so long as the water vapor quantity is not so much that you wet your walls.
Hmmm, what if I could have a metal shop make me a ring, 8.50" open in the center to place the pot in the middle and 11" at the exterior? It would probably cost more than 3 pieces of induction cookware. I found an online fabricator, www.stainlesssteelsupply.com, to see what the cost might be. I really don't want to store and use such large pots if I can avoid it.
This post was edited by Texasgal47 on Thu, Jun 27, 13 at 21:57
They sell induction plates which are used to make non magnetic pots use-able on induction hobs. I can't think of the term for them at the moment. Unfortunately, they are not very successful.
For that reason, I doubt that a home-made metal plate would work well enough. Amazon reviews would be informative.
And it is unknown whether either of these would stop the buzzing.


text:
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