Kilmarnock resume domestic duties against Gretna tomorrow with afar stronger side thanks to the international break.
Manager Jim Jefferies had been facing an injury crisis, with GaryWales, Danny Invincibile, Eric Skora definitely out, and RyanO'Leary, James Fowler and Craig Bryson all struggling.
But Jefferies …
How to Get Married -- After 35
THEIR EYES MET ACROSS A crowded room. It was love at first sight. After a whirlwind courtship and a fairy-tale wedding, they lived happily ever after -- NOT!
Not in real life, at any rate.
Chance encounters, love at first sight, romance -- these may be the elements for a successful soap opera, but not for a successful marriage.
Just look at the high divorce rates. Men and women today find it difficult to stay married, and even to get married.
In Jewish tradition, the question is asked, "What has God been doing since He finished creating the world?"
The answer: "Making marriages."
And this is no easy …
A private security official says gunmen in Nigeria's southern oil region have hijacked an oil-services boat with 13 people onboard, including five expatriate workers.
The official spoke on condition of anonymity due to company prohibitions on speaking to the media. The official said the Tuesday hijacking took place on a river in the southern Niger Delta …
SHEPHERDSTOWN - Unless something is done soon, bats will go theway of the passenger pigeon.
This time it won't be shotguns causing their possible demise, buta nasty fungus called White-nose Syndrome that is killing bats bythe millions, mostly in the caves where they hibernate, scientistssay.
The name comes from a white powder that ends up on the muzzles ofthe animals.
The source of the disease is still unknown, scientists said.
Volunteers, mostly in eastern West Virginia, including theEastern Panhandle, conducted acoustical surveys in an effort toestimate bat populations. The surveys were done from mid-Julythrough Aug. 15.
"Bats," a …
The importance of humane treatment of horses will be stressed onSaturday when horse expert Monty Roberts demonstrates his techniquesat the Fox Valley Ice Arena in Geneva.
Roberts will show off his technique used to train a young horsethat has …
MANAMA, Bahrain (AP) — Proposed reforms submitted to Bahrain's king Thursday seek to strengthen the power of parliament's lower house, the country's only elected body, after political unrest swept the Gulf island nation earlier this year.
The recommendations were drawn up by a government-sanctioned national dialogue committee created to address grievances that emerged during widespread anti-government protests that broke out in February after uprisings elsewhere in the Arab world.
The talks were supported by the United States, which uses Bahrain as the base for the Navy's 5th Fleet, Washington's main counterweight against Iran's influence in the oil-rich Gulf.
However, …
Africa's main development bank says the continent is making a "spectacular" recovery from the global recession.
African Development Bank chief economist Mthuli Ncube said Tuesday the bank's annual economic forecast predicts growth for the continent of 4.5 percent this year and over 5 percent next year. It is then expected to return to the average of about 6 percent it enjoyed …
Pine beetle infestation could kill more than just trees, as safety groups express concern about ramped up production and hauling.
There's a crisis occurring in the BC Interior and according to safety advocates, it's only a matter of time before truckers begin dying as a result. MaryAnne Arcand, manager of the BC Forest Safety Council's TruckSafe initiative, predicted last fall that this would be one of the deadliest hauling seasons on BC logging roads. That didn't materialize this winter, but she says we can thank Mother Nature for most of that.
"We're so thankful. The main factor has been the weather. There have been lots of clear days so we haven't had the usual whiteouts. …
Lindsay Lohan says she was sober before a 2005 car crash near Beverly Hills.
Lohan is being sued for negligence over the accident in which her Mercedes hit a van and injured the driver, Raymundo Ortega.
Ortega, a busboy, alleges Lohan had been drinking at a restaurant called The Ivy before the crash, even though she was underage at the time. Ortega is seeking at least $200,000 (euro138,908) in damages from the 21-year-old actress and the restaurant.
But in a Dec. 15 declaration, Lohan stated: "I did not consume an alcoholic beverage or any type of medication or drug" either before or during her restaurant visit.
A call to …
''We took advantage of their mistakes, and they did the same,'' Fremd coach Chris Piggott said. ''Ours came at a pretty opportune time. I was proud of the guys for not hanging their heads.''
Clint Terry (3-2) went the distance, allowing five hits with nine strikeouts to earn the win. He also drove in a run in the first inning.
Vince Addison and …
Boise is a big bike town but that doesn't mean it's a two-wheeled free-for-all on the roads. Mind your P's and Q's and keep yourself and everyone else safe-and spare yourself a citation.
HARASSMENT OF CYCLISTS AND PEDESTRIANS:
It is a misdemeanor for any person to intentionally cause harm to a pedestrian or cyclist. Whether it is verbal harassment, intimidation or the physical hurling of objects, offenders face a $300 fine and/or six months in jail.
RIDING ON SIDEWALKS AND CROSSWALKS:
Cyclists can ride on sidewalks or in crosswalks so long as it isn't a threat to pedestrians or motorists, unless it is prohibited by a traffic sign. A cyclist must give the …
NEW YORK (AP) — Stocks appear headed to a mixed opening Tuesday following a strong three-day rally.
A day with little change for stocks has been rare so far in March. The Dow Jones industrial average has swung by at least 100 points in four of the last five trading days. Fears about Japan's nuclear crisis and violence in Libya have driven the volatility.
Japanese stocks jumped higher on Tuesday, their first day of trading for the week after a holiday on Monday. The Nikkei 225 index rose 4.4 percent to 9,608.32. European stock markets, which open after those in Asia and before the U.S market, were mixed.
Crude oil prices, a major source of volatility for the market since …
A Marine riding in a Humvee during a shooting that left as many as 19 Afghan civilians dead testified Wednesday that his convoy was fired upon at least three times after it was attacked by a car bomb.
Sgt. Brett Hayes said the gunner in his vehicle was knocked out of the turret by the blast. The gunner returned to his position and began firing, shouting he was being shot at by small arms fire from both sides of the road near a bridge over a dry riverbed.
"I started hearing fire come at us," Hayes said during a fact-finding hearing at Camp Lejeune, adding that he heard small arms fire from AK-47 rifles and cracks of the bullets passing overhead.
Hayes recalled the March 4 attack during the second day of testimony at a rarely used fact-finding proceeding that is investigating the conduct of two officers involved in the shooting.
On Tuesday, defense lawyers presented photographs of men with rifles standing in the riverbed and said the bombing was a well-planned attack on the patrol.
Afghanistan's Independent Human Rights Commission issued a report after the shooting that accused the Marines of firing indiscriminately at pedestrians and people in cars, buses and taxis at six locations along a 10-mile stretch of road. Defense lawyers have maintained the shooting was justified and wasn't indiscriminate.
The administrative Court of Inquiry, scheduled to last two weeks, will recommend whether the officers _ Maj. Fred C. Galvin, 38, commander of the 120-person special operations company, and Capt. Vincent J. Noble, 29, a platoon leader _ should be charged with a crime. That decision will be made by Lt. Gen. Samuel Helland, commander of U.S. Marine Forces Central Command.
Some Marines in the six-vehicle convoy opened fire along a crowded roadway in Afghanistan's Nangahar province after an explosives-rigged minivan crashed into their vehicles. One Marine was wounded. Although an Army investigation concluded 19 Afghan civilians died and 50 were wounded, attorneys for the two officers argue the death toll was lower.
Hayes said that after his Humvee began to roll after the bombing, the vehicle was fired on again from the right side of the road. The gunner fired back, then fired once or twice at a 45-degree angle toward the road, Hayes said, but he didn't know the intended target.
At that point, the gunner realized he had been hit in the arm with shrapnel and Hayes got into the turret for the trip back to base. Hayes said he didn't fire the weapon.
Hayes said the explosion of the vehicle bomb was close enough that he could feel its heat.
The company was on its first deployment after the 2006 creation of the Marine Special Operations Command. After the shooting, eight Marines were sent back to Camp Lejeune and the rest f the company was taken out of Afghanistan.
Maj. Gen. Dennis J. Hejlik, the commander of the Marine Special Operations Command, later said the unit responded appropriately. Marine Corps commandant Gen. James T. Conway also criticized an apology issued by an Army brigade commander, calling it premature because an investigation remained under way.
The Marine Corps last used a Court of Inquiry more than 50 years ago.
___
On the Net:
Marine Special Operations Command: http://www.marsoc.usmc.mil/
Here's a look at the 18 new food categories, which each have their own nutritional criteria, that make up the Smart Choices food labeling program:
_Fresh/frozen/canned fruits and vegetables with no additives
_Processed fruits and vegetables, 100 percent juices
_Breads, grains pasta
_Cereals
_Meat, fish, poultry
_Meat alternatives
_Seeds, nuts, nut butters
_Cheeses
_Milk and dairy products, including soy beverages
_Fats, oils and spreads
_Soups and meal sauces
_Entrees, sandwiches and main dishes
_Meals
_Sauces, dressing and condiments
_Snacks
_Desserts
_Beverages
_Water, plain and carbonated
__
Source: Coalition of food makers, academics, retailers and others led by the Keystone Center.
Boardroom optimism about the economy has plunged to 23% from 67% over the past 12 months and corporate confidence in the Canadian economy is barely registering, according to respondents in the CICA/RBC Business Monitor (Q2 2008) report, a quarterly survey of CAs in executive corporate positions across Canada conducted by the CICA.
It has been a tumultuous 12 months and that is reflected in the drop in confidence and optimism levels of the latest report. "Compared to Q2 2007, which was before the credit crunch and everything else that's gone on in the last year, they're all on the downside," said Kevin Dancey, FCA, CICA president and CEO, of the key confidence and optimism measures. "We'd love to see them back where they were."
The good news for Canadian business is that Canada is experiencing rising export prices coupled with strong domestic demand for goods and services. Some positive effects from an improving US economy are also expected by year's end.
The number of survey respondents indicating that higher energy prices are of major or moderate importance to their company climbed significantly (53% versus 39% in Q2 2007). The widespread impact of gasoline and diesel prices on companies across Canada was evident. Three in four respondents indicated their companies have had to absorb either some (49%) or all (25%) rising energy costs. However, just less than four in 10 respondents in companies facing higher energy costs said they have made operational changes in response; most frequently cited were changes in building operations, shipping/logistics/vehicle usage and general cost reductions.
"With the upward trend in energy costs, sustained energy-cost management is a key focus for all companies," said Dancey. "Well-run enterprises that both recognize the changing environment and control these expenses can achieve a competitive advantage."
The CICA/RBC Business Monitor is issued quarterly and draws upon business insights of CAs in leadership positions in privately and publicly held companies across Canada. This Q2 2008 report, which received national press coverage in both The Globe and Mail and National Post and significant regional coverage, was based on responses to e-mailed surveys completed by 443 CAs of 4,551 identified by CICA as holding senior positions (CFO, CEO and COO).
The CICA/RBC Business Monitor (Q2 2008) is part of an international initiative. The American Institute of Certified Public Accountants in the US and the Institute of Chartered Accountants in England and Wales in the UK also undertake quarterly studies that tap the insights of members in senior positions to provide a barometer of economic activity in their countries and as a basis for future comparative analysis across countries. The CICA/RBC Business Monitor (Q2 2008) is available online at www.cicarbcbusinessmonitor.com.
Single-use membrane chromatography as a polishing option during antibody production is gaining momentum.
ABSTRACT
Monoclonal antibodies (MAbs) continue to dominate the biopharmaceuticals market and their demand is predicted to increase dramatically over the next decade. This demand generates pressure on manufacturers to develop robust and economical large-scale processes that deliver an extremely pure product. Increasingly, manufacturers are turning to disposable chromatography modules for their polishing operations, including membrane adsorbers as an alternative to packed-bed columns. These will significantly reduce downtime by eliminating the need for cleaning and validation. However, economic competition still remains between traditional chromatography and disposable systems. This article discusses the basis of membrane adsorber technology and compares the performance of Q membrane adsorbers and packed-bed columns in terms of contaminant removal, viral clearance, and process economy in antibody manufacture. With new and improved scale-down models, disposable Q membranes now offer competitive advantages over traditional packed-bed systems.
The monoclonal antibody sector is the fastest-growing segment of the biopharmaceuticals market, with a current value of $12 billion, which is expected to increase to at least $20 billion by the year 2010.1 There are currently 23 approved antibodies and antibody-related proteins on the market with hundreds more in preclinical and clinical development.1-3 Despite their hegemonic market position, MAbs are among the most complex and expensive pharmaceutical products to manufacture due to the strict regulatory principles applied to the production of protein-based pharmaceuticals. Over 40% of downstream purification systems are designed for MAbs produced in recombinant mammalian cells.1 Therefore, there is significant interest in finding ways to improve the economy and scalability of downstream processing without compromising purity and reliability.
MAbs represent a special product class within the biopharmaceuticals sector because similar purification steps can be used for all antibodies, regardless of their specificity. This generally involves the use of Protein A affinity chromatography as a primary capture step, because this molecule binds strongly and specifically to the constant region of most IgG class antibodies.4-5 The use of immobilized recombinant Protein A achieves 98% of product-related purity in a single step, significantly reducing the risk of protease contamination, and considerably decreasing the process volume. Thus, all subsequent steps are regarded as polishing operations (Figure 1).6 These downstream steps comprise a mixture of chromatography and microfiltration modules to remove degradation products and any remaining trace contaminants, such as host cell proteins (HCP), nucleic acids, pyrogens, leached Protein A, and viruses.5-7
The most popular choice among the polishing steps in current MAb manufacture is flow-through anion exchange chromatography (FT-AEX). This is because many contaminants, including viruses, nucleic acids, endotoxins, and some HCPs, tend to be negatively charged at neutral pH, and are therefore retained by the resin, whereas antibodies are positively charged under the same conditions and flow through efficiently.8-9 However, a major disadvantage of packed-bed FT-AEX is that to cope with the high-throughput of today's MAb manufacturing processes, the diameter of the columns needs to be very large to facilitate the necessary volumetric flow rates. As the production scale increases, certain capital and operational costs need to be considered (e.g., resin life studies, packing, cleaning validation, and storage validation studies). Thus, alternative polishing technologies become increasingly attractive.8,10-11
Principles of Membrane Chromatography
Unlike traditional column chromatography, which uses packed beads as the resin, membrane chromatography involves the use of thin, synthetic microporous or macroporous membranes that are chemically activated to fulfill the same function. An important mechanical difference between the two processes is that in membrane chromatography, the transport of solutes to their binding sites occurs mainly by convection, whereas pore diffusion is minimal compared with packed beads (Figure 2). This diminishes mass transfer resistance, reducing the time required for adsorption, washing, elution, and regeneration.8 Therefore, membranes potentially allow increased flow rates in large-scale processes, leading to an overall reduction in process time.8,11-12 The comparatively large pore size of membranes provides a higher binding capacity for large biomolecules such as viruses and DNA, even at high flow rates. Another advantage of using disposables is that exhausted membrane modules can be replaced with new ones rather than the traditional requirement to clean the columns, validate the cleaning procedure, and repack with resin. Various approaches for the purification of proteins using resins and membranes have already been reviewed.5,10
Unfortunately, although efficient membrane adsorption has been demonstrated for laboratory and pilot scale protein purification, several limitations remain to be overcome before such devices can be used routinely in processscale operations. Some of these limitations reflect structural artifacts, such as uneven pore-size distribution and variations in membrane thickness, which can be addressed through the use of multiple-layer configurations. However, one attribute that is proving intractable is the low binding capacity of such membranes for smaller molecules compared to column resins, which results from the lower surfaceto-bed volume ratio and flow distribution problems. Q anion exchange adsorber devices have been used for the preparation of vaccines and gene therapy vectors, as well as for endotoxin removal on a process scale.13-18 The low binding capacity is a serious disadvantage when using membranes in retention mode, but this problem can largely be ignored for processes where the flow-through mode is used. Flowthrough Q membrane adsorbers therefore provide an ideal solution to the requirement for a robust, highthroughput polishing step in antibody production, removing impurities, and including viruses, which are present at less than 1% concentration.10-11
Performance Comparisons
Several companies have carried out evaluation studies to compare the performance of membrane adsorbers and traditional packed-bed columns, demonstrating the competitive nature of Q membranes compared to Q columns. The standards used to measure the performance of each system are not directly comparable and care needs to be taken when reviewing the performance data. One important aspect is the measurement of process capacity, which in the case of column chromatography is usually expressed as grams of product per liter of resin, with a typical value of 50-100 g/L. A direct comparison between packed-bed columns and membrane adsorbers means comparing the volume of membrane to the volume of resin, even though the volume of a membrane must take into account its thickness, which has little impact on binding capacity and can be difficult to calculate accurately due to uneven thickness within a membrane sheet. Membrane vendors use the area of membrane to express process capacity, and also sell membranes by unit area rather than by unit volume. Therefore, it is more convenient and appropriate to express process capacity in grams of product per square meter of membrane.10-11
Since one of the great advantages of membrane adsorbers is fast processing time, flow rate is also an important comparator between column and membrane modules. In the case of columns, flow rate is calculated as linear velocity (cm/h), whereas several different units have been used for membrane chromatography; each is based on flux rate, analogous to the system used in cross-flow filtration. As for process capacity measurements, flow rate measurements such as membrane volume per minute and device volume per minute depend on membrane thickness. Therefore, it is able to measure flow rate as a linear velocity, calculated according to the cross-sectional area of the membrane.10-11 The maximum linear flow rate reported for packed-bed columns in process-scale AEX procedures is about 100-200 cm/h using Q-Sepharose Fast Flow (GE Healthcare).10 In contrast, linear flow rates of up to 600 cm/h can be achieved with functionally equivalent Q membranes. This occurs with no compromise in efficiency, since the large membrane pores provide a high binding capacity for viruses and nucleic acids even at these high flow rates. Another advantage is that much less buffer is used with membrane chromatography compared to conventional packed-bed chromatography.
A number of studies published since 2001 have demonstrated the potential of Q membranes for largescale processes. In the first such study, a 10-layer Sartobind Q membranestacked Swinny 13-mm-folder scaledown model (Sartorius AG) for an antibody process showed that the Sartobind Q membrane provided a 2.3 log reduction value (LRV) for murine leukemia virus (MuLV) at a flow rate of 620 cm/h and a capacity of 2000 g/L (or about 550 g/m^sup 2^).8 The LRV decreased as the antibody load increased. For example, 1.0 LRV was achieved at a capacity of 1100 g/m^sup 2^, 0.5 LRV was obtained at a capacity of 1650 g/m^sup 2^, and 0.3 LRV was reported at a capacity of 2755 g/m^sup 2^. In contrast, Q-Sepharose Fast Flow provided a viral clearance power of >5.1 LRV at a capacity of 50 g/L.
In a similar study, a Sartobind Q capsule module (Q-10 in.) was used for antibody production and a Sartobind Q 75 15-layer module was used for viral clearance during large-scale antibody production.19 The capacity was estimated at 450 g/m^sup 2^ with better viral clearance rates than described above. Approximately 4.0 LRV was reported for respiratory enteric orphan virus III (Reo-3), MuLV, and pseudorabies virus (PRV).
More recently, the use of Sartobind Q in a 15-layer format was reported in an antibody pilot plant producing material for a toxicity study.12,20 The scale-down model used the Sartobind Q75. The detailed study demonstrated that Sartobind Q75 has the capacity to remove HCP and viruses at a neutral pH and conductivity below 3 mS/cm. Under these conditions, the virus clearance results were excellent: 5.57 LRV for MuLV, 7.28 LRV for Reo3, 6.77 LRV for minute virus of mice (MVM), and 5.67 LRV for PRV.12,20 However, the process capacity was estimated at 480 g/m^sup 2^ or 1750 g/L membrane volume.
The performance studies outlined above suggest that the process capacity of Q membranes (up to 2 kg/L or 550 g/m^sup 2^) is significantly higher than that of Q resin (up to 100 g/L). From an economic perspective, however, this comparison does not take into account the relative cost of the media.10 A direct comparison of resins and membranes based on volume show that membranes are on average 15 times more expensive, and are in any case priced by area rather than volume. Ultimately, cost analysis indicates membrane adsorber process capacity needs to exceed 2 kg/m^sup 2^ or 8 kg/L to compete with packed-bed resins (i.e., a fourfold increase), as explained below.
Cost Analysis with the Original Scale-Down Model
The original scale-down model process capacity indicates that Q membrane chromatography is impressive in performance but economically unfeasible. To extend that analysis, consider a cost comparison between Q-Sepharose Fast Flow and Sartobind Q using a throughput of 13.5 kg of antibody, and assuming a water-for-injection (WFI) cost of $3/L (Table 1). The cost of 1 m^sup 2^ of membrane is about $2,400 but this would drop to approximately $2,000 at larger scales. The device is disposable and not reusable. Although this saves on cleaning and reuse validation, it does increase the raw material cost. To process 13.5 kg of antibody, the cost of membrane alone is about $54,000 at a loading capacity of 500 g/m2. This, together with the labor cost of buffers, generates a total cost for each cycle to be $58,588. The process capacity of Q-Sepharose Fast Flow commonly used for antibody purification is about 70 g/L. Thus, at $500/L, the initial cost is about $110,000 for 220 L of resin, which would be packed into a 100-cm column to a bed height of 25 cm. The Q resin can be reused for at least 100 cycles, thus, the resin cost per cycle is only $110,010. However, the cost of the resin is only one part of the true overall cost of the unit operation. Additional costs include labor and buffers (equilibration, wash, regeneration, and storage). The total cost is approximately $18,500 per cycle. Thus, the Q column unit operation is more cost-effective than the Q membrane unit operation at a process capacity of 500 g/m^sup 2^.
A careful comparison of the items listed in Table 1 shows how we arrive at the target process capacity of 2 kg/m^sup 2^ required to make Q membranes competitive with resins in a large-scale process. At this capacity, the total cost for one cycle, including the Q membrane ($13,500), instillation ($700), equilibration buffer ($1820), and wash buffer ($620), is about $16,640. This makes the membrane adsorber very competitive against the column unit operation cost per cycle of $18,500. This comparison does not include the column packing and validation costs incurred with resin reuse. Therefore, it is necessary to demonstrate a process capacity of 2 kg/m^sup 2^ on a large scale and validate this with a scale-down model while still achieving excellent quality parameters such as DNA and viral clearance.
A New Scale-down Model
Currently, scale-down models (including Sartobind Q75) are not suitable for viral clearance studies because of process capacity limitations generated by operational backpressure with the scale-down devices. The liquid flow path in scale-down models is not the same as that found in the large-scale capsule, and thus tends to generate extremely high operational backpressure when a high flow rate is applied (450 cm/h). The scale-down model, such as Sartobind Q75, is made with stacks of several flat sheets, and the axially directed velocity of a mobile solution is much faster in the center of the membrane unit than near the edges of the adsorptive bed.10-11 In contrast, radial flow adsorbers, prepared by spirally winding a flat sheet membrane over a porous cylindrical core, are used in a capsule for large-scale processes.10 Distorted or poor inlet flow distribution has less of an impact in the scaled-up capsule, thus leading to a smaller operational backpressure drop for large-scale operations.10
Scalability and operating backpressure are major issues for the membrane absorber scale-down model. Extremely high operational backpressure in the scale-down model reduces the capacity considerably, resulting in the use of an oversized membrane at process scale. This may cause errors in economic calculations and undervalue the economic benefits of membrane adsorbers. Large-scale membranes do not have backpressure issues. However, the use of large membranes in a scale-down model is also impractical for viral clearance studies because of the high cost of viruses and feedstock.
If Q membrane adsorbers are made from diethylaminoethyl (DE) cellulose material, the conductivity of the operation solution directly affects operational backpressure. It is also important to determine the effects of operation temperature on membrane backpressure drop because a relatively low operation temperature would increase the viscosity of an antibody solution. In addition, some buffers are temperature-sensitive. At a low temperature, the low conductivity can generate a high operational backpressure leading to a low process capacity. These factors have necessitated a redesign of the scale-down model.21
The new scale-down model mimics the liquid flow path found in the large-scale modules. When the newly designed Q125 was examined at a flow rate of 450 cm/h with a process capacity of 3 kg/m^sup 2^, excellent viral clearance was achieved with four model viruses (Table 2). A maximum operational backpressure of <16 psi was observed. This new device also achieves performance parameters comparable to those of the larger Sartobind Q units, with a process capacity of >3.6 kg/m^sup 2^ (total membrane surface) or >13.2 kg/L (membrane volume) at a maximal flow rate of 600 cm/h with an operational backpressure of <18 psi. A LRV of >5.6 was achieved for MuLV21. Since such high process capacity required a significant amount of feedstock and virus, the clearance studies with PRV, MVM and Reo-3 viruses were not carried out. The clearance studies with the four model viruses were run at a flow rate of 450 cm/h and process capacity of 3 kg/m^sup 2^.
The fact that the viral clearance performance at high flow rate and high capacity is similar to the MuLV LRV at 450 cm/h and a capacity of 3 kg/m^sup 2^ suggests that a similar viral clearance value could be achieved at a higher operational flow rate and capacity for the other three model viruses.
Mass balance data in a viral clearance study is extremely important to demonstrate efficient virus removal by a membrane adsorber device. For example, 100% recovery was obtained for PRV (n = 2), Reo-3 (n = 2) and MVM (n = 2) virus particles with particle sizes of 18-200 nm when the membrane was stripped with 1 M NaCl, demonstrating efficient charge capture for the three model viruses. When the membrane was treated with high salt, an average of only 70% viral recovery (n = 3) was achieved for MuLV. This may reflect the possibility that 1 M NaCl inactivated some MuLV particles, they irreversibly bound to the membrane or were inactivated by mechanical stress during the process of adsorption and desorption.
These concepts and parameters have been successfully applied to purify four lots of recombinant human antibody at the 2000-L scale. The impurities, including HCP, DNA, and leachable rProtein A, were below the limit of detection following Q membrane purification. Figure 3 shows the typical MVM viral clearance power when the Sartobind Q 125 device is used. These data support the replacement of Q packedbed columns with membrane adsorbers as a polishing step in flow-through chromatography for process-scale antibody production.
Cost Analysis with the New Scale-down Model
At a process capacity of 3 kg/m^sup 2^, Q membrane chromatography is very competitive with resin chromatography when using $3/L as the cost of WFI in the calculations (Table 1). Although this is the cost currently utilized by contract manufacturers, the estimated WFI costs for large biotech firms can be as low as $0.2/L Table 3 compares the total cost for membrane operation at a capacity of 3 kg/m^sup 2^ versus Q column operation at a capacity of 70 g/L using only $0.2/L for WFI cost. In this scenario, column unit operation is more cost efficient ($9,168 per cycle) than membrane unit operation ($11,738) even at the high process capacity. The membrane price dropped only $400 with the decrease in WFI cost whereas the column saved more than $9,000. This highlights the difficulty in comparing the real cost of a unit operation and the danger of minimizing something as common as WFI. This calculation also did not take into account the cost of tanks to contain the extra buffer needed for the column.10
For commercial manufacturing, column chromatography has a supplemental upfront cost that can be grouped into three main categories: development, manufacturing, and validation costs. Development and validation are performed once for the Q column, and these studies are not needed subsequently as long as the columns are run in exactly the same format, including bed width and height, buffers, and operation conditions such as flow rate and cleaning-in-place. Upfront manufacturing costs include column hardware and packing equipment. In the current work, 40 lots per year over a 10-year production time were used for the comparison between Q columns and Q membranes. This is a reasonable time period given product patents and process changes. The 40 lots per year are designed for production efficiency or product demand and four columns are required for the 10-year production based on 100 runs (cycles) per column. Obviously, some products are run more frequently and some less frequently. If more cycles are run between process changes, the initial investment would be averaged over more runs.10
For a ten-year production time or 400 cycles, development activities are not required for membrane chromatography and about 1,800 m^sup 2^ of Q membrane is required based on a process capacity of 3 kg/m^sup 2^. This disposable system does not incur most of the initial investment costs listed above because the membrane is single use only. Moreover, the validation cost is minimal and no packing studies are required. After summarizing all the costs for column chromatography such as resins, buffers, labor, and validation studies, industry has the potential to spend more money on column operations than Q membrane operations.10
Potential Limiting Factors of Membrane Adsorbers
Even with improved scale-down devices, the operational backpressure for Q membrane chromatography will always influence process capacity because of the high viscosity of the solution. However, viscosity depends on factors such as operational temperature, pH, conductivity, and protein concentration if the solution contains protein.
Temperature is likely to be the most important factor because viscosity increases as the temperature falls, and low temperatures are preferred to maintain product stability. The viscosity of an antibody solution is higher than the viscosity of the control buffer solution at the same low temperature. At 4�C, the viscosity of an antibody solution (3 mg/mL at pH 7.2 and about 4 mS/cm) is much higher than at 8�C or at 13�C. 21 High viscosity at low temperature significantly increases operational backpressure and results in lower flow rates. At a low temperature, even smaller particles can generate significant backpressure. Indeed, at room temperature, the operational backpressure is independent of virus particle size (a similar backpressure curve is found for Reo-3, PRV, and MuLV particles, which range in size from 18-200 nm). Although a low operational temperature reduces membrane process capacity because of the higher operational backpressure created by the higher viscosity of protein and buffer solutions, our data suggest that the low temperatures have very minimal impact on viral clearance with the backpressure.21
Another limiting factor is found when the buffer pH is close to the pI value of protein. The viscosity appears to peak at the pI of the molecule studied.21 Other limiting factors, including conductivity, need to be monitored. Low conductivity causes more molecular interaction to occur between antibodies, thereby increasing the solution viscosity.
Conclusions
The use of disposable Q membrane chromatography modules is becoming increasingly attractive in largescale antibody processing as a result of impressive performance and economic comparisons with traditional anion-exchange chromatography. Membranes benefit from faster processing times due to convective flow, higher process capacity, and up to a 95% reduction in buffer use, all helping to reduce costs in head-to-head comparisons. Single-use membrane chromatography is robust and simple to use, with no column packing or subsequent cleaning validation required. In particular, the Q membrane is an effective and robust unit operation to remove viruses.21 Q membranes have a relatively short history and we can anticipate further improvements with a new generation of even smaller scale-down models showing better host-cell protein removal capacity. We conclude that Q membrane chromatography in flowthrough mode is a viable alternative to Q column chromatography as a polishing step for late-stage processscale antibody production.
[Sidebar]
A benefit of using disposables is that exhausted membrane modules can be replaced with new ones instead of cleaning the columns.
[Sidebar]
Because one of the great advantages of membrane adsorbers is the fast processing time, flow rate is an important comparator between column and membrane modules.
[Sidebar]
Mass balance data in a viral clearance study are extremely important to demonstrate efficient virus removal by a membrane adsorber device.
[Sidebar]
Single-use membrane chromatography is robust and simple to use, with no column packing or subsequent cleaning validation required.
[Reference]
References:
1. Daemmrich A, Browden ME. A rising drug industry. Chem Eng News. 2005; 6:28-42.
2. Walsh G. Biopharmaceuticals: approvals and approval trends in 2004. BioPharm Int. 2005; 18(10): 58-65.
3. Chadd HE, Chamow SM. Therapeutic antibody expression technology. Curr Opin Biotechnol. 2001; 12:188-194.
4. Fahrner RL, Whitney DH, Vanderlaan M, Blank GS. Performance comparison of protein A affinity-chromatography sorbents for purifying recombinant monoclonal antibodies. Biotechnol Appl Biochem. 1999; 30(2):121-8.
5. Fahrner RL. Industrial purification of pharmaceutical antibodies: development, operation, and validation of chromatography process. Biotechnol Gen Eng Rev. 2001; 18:301-327.
6. Zhou J, Tressel T, Hong T, Solamo F, Dermawan S. Utilize current separation technology to achieve a cost-effective robust process for recombinant monoclonal antibody production. Antibody World Summit. Jersey City, NJ; 2005.
7. Tressel T. Development of a generic platform and use of statistically designed experiments to enable rapid development of several antibodies and increase throughput for first in human antibodies. Antibody production and downstream processing, IBC conference. San Diego, CA; 2004.
8. Knudsen H, Fahrner R, Xu Y, Norling L, Blank G. Membrane ion-exchange chromatography for process-scale antibody purification. J Chrom A. 2001; 907:145-154.
9. Kemp G, O'Neil P Large-scale production of therapeutic antibodies: considerations for optimizing product capture and purification. Antibodies (Subramanian G, Ed.). 2004; 1.2 vols, Plenum Publishers, New York.
10. Zhou J, Tressel T. Basic concepts in Q membrane chromatography for largescale antibody production. Biotechnol Prog. 2006; 22:341-349.
11. Zhou J, Tressel T. Q membrane chromatography as a robust purification unit for large-scale antibody production. Bioprocess Int. 2005; 3:32-37.
12. Zhang R, Bouamama T, Tabur P, Zapata GA. Q membrane chromatography application for human antibody purification process. BioProduction. 2004; http://www.abgenix.com/ documents/ Bioproduction2004. pdf.
13. Deshmukh RR, et al. Large-scale purification of antisense oligonucleotides by high-performance membrane adsorber chromatography. J Chrom A. 2000; 890:179-92.
14. Grunwald AG, Shields MS. Plasmid purification using membrane-based anion-exchange chromatography. Anal Biochem. 2001; 296:138-41.
15. Zhang S, Krivosheyeva A, Nochumson S. Large-scale capture and partial purification of plasmid DNA using anion-exchange membrane capsules. Biotechnol Appl Biochem. 2003; 37: 245-9.
16. Teeters MA, Conrardy SE, Thomas BL, Root TW, Lightfoot EN. Adsorptive membrane chromatography for purification of plasmid DNA. J Chrom A. 2003; 989:165-73.
17. Kuiper M, Sanches RM, Walford JA, Slater NK. Purification of a functional gene therapy vector derived from moloney murine leukaemia virus using membrane filtration and ceramic hydroxyapatite chromatography. Biotechnol Bioeng. 2002: 80:445-53.
18. Specht R, et al. Densonucleosis virus purification by ion exchange membranes. Biotechnol Bioeng. 2004; 88:465-73.
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[Author Affiliation]
JOE X ZHOU is principal scientist, PPD, TIM TRESSEL is director, PPD, SAM GUHAN is executive director, process and analytical sciences, at Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA, USA. 805.313.4065. joez@amgen.com.
The judge in the R. Kelly child pornography trial is threatening to issue a warrant for the arrest of a Chicago Sun-Times reporter.
Reporter Jim DeRogatis (day-roh-GAH'-tus) got hold of the sex tape at the heart of the case in 2002. He didn't appear for a morning hearing regarding his testimony at Kelly's trial.
An angry Judge Vincent Gaughan (GAWN) told a Sun-Times attorney he'd give him 15 minutes to find something in Illinois law that gives DeRogotis the right to defy an order to appear in court.
Gaughan has rejected arguments that the reporter's actions should be privileged under the First Amendment and that he shouldn't have to appear.
Prosecutors rested their case Monday, and jurors have the day off.
The singer has pleaded not guilty.
Manchester United returned to the Premier League summit by routing bottom-place Portsmouth 5-0 on Saturday, while Manchester City lost 2-1 at relegation-threatened Hull.
Wayne Rooney and Dimitar Berbatov each scored for United, while three Portsmouth players scored own goals to add to the debt-ridden club's woes.
United will now hope Chelsea is beaten by third-place Arsenal on Sunday so the champions retain their one-point lead.
The top four regained a familiar look after Liverpool climbed to fourth with a 1-0 victory over Everton, while Tottenham drew 0-0 with Aston Villa and Man City lost.
At Old Trafford, Wayne Rooney headed in from close range from Darren Fletcher's cross and Portsmouth defender Anthony Vanden Borre turned in Nani's attempted cross just before halftime.
After the break, Michael Carrick's shot deflected in off Richard Hughes before Dimitar Berbatov tormented the Pompey defense and fired home the fourth. Mark Wilson became the third Pompey player to score an own goal to round off the miserable day for the visitors.
United's crosstown rival City slumped to its second league loss since Roberto Mancini replaced Mark Hughes as manager in December as Hull climbed out of the relegation zone.
United States forward Jozy Altidore scored his first Premier League goal at his 18th game and George Boateng volleyed in Hull's second. Emmanuel Adebayor's goal for City wasn't enough to spark a comeback, despite Patrick Viera coming on as a substitute to make his debut for the club.
"In the first half we didn't play very well. Hull were more aggressive and put us under pressure," Mancini said. "In the second half we played very well, for me. When we brought on Patrick Vieira and Adam Johnson, the game changed. If we play like we did in the second half, we can arrive in the top four _ but not if we play like we did in the first."
At White Hart Lane, Spurs were angered by their failure to get a penalty when Stiliyan Petrov appeared to trip Jermain Defoe in the closing minutes.
"It looked like a penalty, but we got a good decision and we're happy," Villa defender Richard Dunne said.
Tottenham's failure to find a way past Villa goalkeeper Brad Friedel at White Hart Lane meant Liverpool's lunchtime victory kept the 18-time champions in fourth.
Following a lackluster start to the season, Liverpool has now collected 17 points from a possible 21 in the last seven matches.
Despite Everton defender Phil Neville clinging to him, Dirk Kuyt sent Liverpool captain Steven Gerrard's inswinging corner past goalkeeper Tim Howard from close range in the 55th minute for the game's only goal.
Liverpool was a man down from the 35th after Sotirios Kyrgiakos was sent off for a two-footed challenge on Everton midfielder Marouane Fellaini, who escaped punishment for stamping on the Greek defender in the same incident.
Everton winger Steven Pienaar was shown a second yellow card in the 90th for a light challenge on Gerrard, but the South African was fortunate not to be sent off in the first half for a foul on Javier Mascherano.
"Derbies are all about passion, commitment and fight, and we've beaten a good Everton side here with 10 men, so we should be proud and enjoy this," Gerrard said. "Our attitude prevailed, we stuck together and then took our chance when it came."
Burnley climbed out of the relegation zone and sent West Ham back into the bottom three with a 2-1 victory over the east London club.
Bolton is just one point above West Ham after drawing 0-0 with Fulham and Wigan is a further point ahead after holding Sunderland to a 1-1 draw.
Japan's opposition-controlled upper house of parliament rejected the government's nominee for Bank of Japan chief Wednesday.
The opposition had threatened for days to block the appointment the Toshiro Muto, accusing the government of using strong-arm tactics to push through its personnel decisions. Muto, 64, is currently a central bank deputy governor and was nominated for the top post last week.
The term of the current Bank of Japan governor ends March 19. The candidates for governor and the two deputy governors at Japan's central bank need approval from both houses of parliament.
The ruling coalition controls the more powerful lower house, and has used that edge recently to ram through some bills that require approval from only that house.
The stalemate in legislature over who will head the central bank of the world's second largest economy is a major embarrassment for Japan's government at a time when fears are growing about a global slowdown.
The upper house voted to oppose Muto's appointment in a vote of 129-106.
In a twist, the opposition approved one of the government appointments for deputy, former BOJ Executive Director Masaaki Shirakawa, 58, now a Kyoto University professor, who won on a vote of 230-7.
The nominee for the other deputy post _ Takatoshi Ito, 57, a professor at University of Tokyo _ was voted down along with Muto.
NEW YORK (AP) — The euro rose against the dollar for the second day in a row on hopes that European leaders will come up with a solution to deal with the region's growing debt crisis.
Finance ministers from the 17 countries that use the euro met in Brussels Tuesday to discuss ideas. The finance ministers are considering a number of plans for protecting the region's shared currency from collapse. One idea being floated is having countries relinquish some control over their finances to a central European authority.
Early in the meeting they approved the next $10.7 billion installment of Greece's bailout loan. Without that money, the country would have run out of cash next month. European Union finance ministers are also expected to meet on Wednesday.
Pressure to come up with a solution has escalated in the past couple of weeks as borrowing costs have risen in Italy, Spain and even Europe's biggest economy Germany. Rising borrowing costs are a sign that investors think the country may not be able to pay its debts.
The euro rose to $1.3331 late Tuesday from 1.3306 late Monday. The euro has fallen nearly 4 percent so far this month on fears that Europe's debt crisis would spread to other countries.
In other trading, the British pound rose to $1.5607 from $1.5496. The dollar fell to 77.93 Japanese yen from 77.99 Japanese yen, to 0.9203 Swiss franc from 0.9234 Swiss franc and to 1.0301 Canadian dollar from 1.0366 Canadian dollar.
ANN ARBOR, Mich. - Bo Schembechler, who became one of college football's great coaches in two decades at Michigan, died Friday after taping a TV show on the eve of the Wolverines' No. 1 vs. No. 2 showdown with perennial rival Ohio State. He was 77.
Schembechler collapsed during the taping of a television show in Southfield and was taken by ambulance to a nearby hospital. His death at 11:42 a.m. was confirmed by Mike Dowd, chief investigator for the medical examiner's office in Oakland County.
Schembechler became ill at the studios at WXYZ-TV in Southfield, the station said. He also became ill there and was hospitalized Oct. 20.
Police were sent to the station around 9:25 a.m. along with the city's fire department and escorted an ambulance to Providence Hospital, Southfield police spokesman John Harris said.
Schembechler met with the media earlier this week to discuss Saturday's big game.
During the news conference, he discussed the device that was implanted to regulate his heartbeat after he was hospitalized last month.
He said the device covered about half his chest and that doctors still were adjusting it.
Schembechler said he did not plan to attend the game in Columbus, Ohio, and that he didn't attend road games anymore.
Schembechler had a heart attack on the eve of his first Rose Bowl in 1970 and another one in 1987. He has had two quadruple heart-bypass operations.
The seven-time Big Ten coach of the year compiled a 194-48-5 record at Michigan from 1969-89. Schembechler's record in 26 years of coaching was 234-64-8.
Schembechler's Wolverines were 11-9-1 against the Buckeyes. But fans in both states generally agree that the rivalry's prime years were 1969-78, when Schembechler opposed his friend and coaching guru, Woody Hayes. Ohio State prevailed in those meetings, going 5-4-1.
"It was a very personal rivalry," Earle Bruce, who succeeded Hayes as coach, once said. "And for the first and only time, it was as much about the coaches as it was about the game.
"Bo and Woody were very close because Bo played for Woody at Miami of Ohio, then coached with him at Ohio State. But their friendship was put on hold when Bo took the Michigan job because it was the protege against mentor."
Thirteen of Schembechler's Michigan teams either won or shared the Big Ten championship. Fifteen of them finished in The Associated Press Top 10, with the 1985 team finishing No. 2.
Seventeen of Schembechler's 21 Michigan teams earned bowl berths. Despite a .796 regular-season winning percentage, his record in bowls was a disappointing 5-12, including 2-8 in Rose Bowls.
The mythical national championship eluded Schembechler, but he said that never bothered him.
"If you think my career has been a failure because I have never won a national title, you have another thing coming," Schembechler said a few weeks before coaching his final game. "I have never played a game for the national title. Our goals always have been to win the Big Ten title and the Rose Bowl. If we do that, then we consider it a successful season."
His last game as Wolverines coach was a 17-10 loss to Southern California in the 1990 Rose Bowl. One week later, Schembechler - who also had been serving as Michigan athletic director since July 1988 - was named president of the Detroit Tigers.
OSTERSUND, Sweden (AP) — Martin Fourcade of France outpaced skiers from the Czech Republic and Germany to win the 20-kilometer biathlon in the season-opening World Cup on Tuesday.
Fourcade missed only one target on the course in Ostersund to cross the line comfortably in 53 minutes, 29.8 seconds.
The closest rival, 1:54.3 behind, was Cezch Michal Slesingr, who also missed one target.
Germany's Simon Schempp finished third, 1:54.5 back.
Last year's overall cup winner, Norwegian Tarjei Boe missed four targets to place 25th, 3:59.5 off the pace.
To make sense of complex biological systems, biophysicists often devise methods to reduce the number of interacting components so that underlying molecular mechanisms are revealed: The ultimate form of this reductionist approach is to work with single molecules. On the other hand, physiologists usually prefer to work with intact isolated systems that closely mimic live conditions. To integrate information obtained by both approaches it is useful to have techniques that bridge the gap between "physiology" and "molecular biophysics". Suzuki, Fujita, and Ishiwata (in this issue) report a new technique to study the mechanism of force generation in a semi-intact muscle preparation. Their experiment involves threading an individual actin filament into a chemically treated muscle myofibril so that the forces between native myosin filaments and the single actin filament can be measured. The muscle lattice structure is preserved and the experimental conditions are close to physiological. What can we learn from such a new approach and how will it help answer some of the thorny issues surrounding the detailed mechanism of force generation by actomyosin in muscle fibers?
We now know that there are at least 18 different myosin families (1) and they are responsible not just for muscle contraction but also for a wide variety of other cell motilities. In recent years, much interest has turned to the newly discovered, nonmuscle myosins. However, the mechanism of muscle contraction remains of central interest to the field and muscle myosin remains the "gold standard" in our quest to understand the detailed mechanism of force generation by actomyosin. In general, we know that force production in muscle is due to the cyclical interaction of myosin heads with actin, coupled to the hydrolysis of ATP to ADP and Pi and this is known as the cross-bridge cycle. We believe that one mechanical "kick" is produced for each ATP molecule consumed.
MUSCLE FIBERS
The near crystalline arrangement of interdigitating thick and thin filaments within the sarcomeres of a muscle cell (or fiber) cause individual myosin motors to work as a team and generate huge forces and rapid velocities of shortening. Early researchers exploited the natural diversity of muscle types present in different organisms. Some muscles are very fast, others are slow, some are adapted for self-sustaining oscillatory contractions, and others lock up in a "catch state" and consume little ATP. Comparative studies using different muscle types have led to important insights into the general contractile mechanism. Using vertebrate striated muscle fibers, we know that a single nerve impulse will activate billions of myosins in concert and that a sudden change in muscle length will partially synchronize the myosin ATPase cycles. In fact, much of what we know about the mechanism of force generation derives from sudden "length-step" experiments made using activated single muscle fibers from frog (2) or rabbit combined with a variety of other, synchronized measurements. One of the beauties of these studies is that they can be performed at close-to-physiological conditions. In recent years, use of synchrotron radiation (3) and fluorescent optical probes (4), together with our knowledge of the actin and myosin atomic structures, has given new insights into the way myosin generates force in muscle.
NOISE ANALYSIS
Part of the central dogma of the crossbridge cycle is that each myosin should work independently and generate force as a square-wave pulse each time it cycles with actin. The amplitude of force fluctuations produced by these stochastic interactions will depend upon the square root of the number of myosins, whereas total force is linear with number. Statistical analysis of noise can give insights into the underlying molecular mechanisms and this approach was first pioneered in nerve research (5). However, because a single muscle fiber contains ~10^sup 12^ molecules the expected fluctuations in force would be just one millionth of the total force. Early attempts to make these very challenging measurements in muscle failed (6,7) and it was only recently that successful noise measurements have been made using single actin filaments (8). Notably, no one has yet measured noise produced by cross-bridge cycling in intact muscle.
SINGLE MOLECULES
Over the past 10 years, single-molecule mechanical studies have enabled direct observation of individual power strokes by single myosin heads (9). In these experiments, an individual actin filament is attached between two microbeads that are held and manipulated using optical tweezers. By position ing the actin close to a single myosin molecule the stochastic mechanical interactions can be measured using a position-sensitive photodetector to monitor the microbead positions. These highly reductionist, single-molecule experiments have given great insight into the mechanism of force generation by actomyosin. The techniques have also been invaluable for studying nonmuscle myosins and the natural variation between myosin families has given new clues about the underlying molecular mechanism of force generation. However, many questions remain about how myosin works in intact muscle fibers and there are still significant gaps in our understanding: For instance, we still need to know more about the role played by muscle structure in the regulation of contraction in striated, smooth, and cardiac muscles; whether individual myosin heads really behave as independent force generators in the intact muscle lattice; what processes are responsible for rapid tension recovery after a muscle length step; and how insect flight muscles are "stretch-activated".
HYBRID SYSTEM
To uncover the secrets that lie hidden within the intricate structure of muscle, Ishiwata's group have developed a hybrid system that lies halfway between muscle-fiber and single-molecule studies. They hold a single actin filament at one end by attaching it specifically to a plastic microbead that is held and manipulated using optical tweezers. They then take a muscle myofibril from which the native actin filaments have been specifically removed (by treatment with gelsolin) and position it so that the actin filament can be threaded into the frayed end of the myosin filament lattice. Since the preparation is immersed in an MgATP-containing solution, the actin is pulled in toward the M-line by the myosin heads. This motion is monitored using a videocamera system. Movement of the actin filament can be arrested and even reversed by adjusting the power of the laser tweezer. By stopping the motion at various predetermined positions, the researchers were able to measure the relationship between force and filament overlap. Their results seem to match the classical results obtained from intact muscle fiber preparations: The average force is directly proportional to overlap between actin and myosin filaments and hence is determined by the number of myosin heads available to bind.
The results so far seem consistent with our classical understanding of the muscle contractile mechanism but the experiment remains tantalizing as the time response of the recording equipment is currently insufficient to analyze the stochastic noise produced by the relatively small number (-100) of myosins. In the future, experiments made with increased time resolution should give important additional information and permit more advanced statistical analysis (10). One hopes that such studies will shed light on some of the mechanistic questions raised earlier. We also eagerly await studies of the diverse muscle types that were used early on in our quest to "solve muscle", for instance, insect flight and scallop muscle fibers. The possibility also exists to mix and match different thin-filament regulatory systems with different myosin thick-filament lattices. The new ability to look at small ensembles of myosins in their native lattice arrangement and also vary the type of actin or myosin used means we can let the frog see the rabbit.
[Sidebar]
doi: 10.1529/biophysj.105.059501
[Reference]
REFERENCES
1. Sellers, J. R. 2000. Myosins: a diverse superfamily. Biochim. Biophys. Acta. 1496: 3-22.
2. Huxley, A. F., and R. M. Simmons. 1971. Proposed mechanism of force generation in muscle. Nature. 233:533-538.
3. Huxley, H. E. 2004. Recent x-ray diffraction studies of muscle contraction and their implications. Philos. Trans. R. Soc. Lond Ser. B. Biol. Sci. 359:1879-1882.
4. Irving, M., T. St Claire-Allen, C. Sabido-David, J. S. Craik, B. Brandmeier, J. Kendrick-Jones, J. E. T. Corrie, D. R. Trentham, and Y. E. Goldman. 1995. Tilting of the light-chain region of myosin during step length changes and active force generation in skeletal muscle. Nature. 375: 688-691.
5. Katz, B., and R. Miledi. 1970. Membrane noise produced by acetylcholine. Nature. 226:962-963.
6. Borejdo, J., and M. F. Morales. 1977. Fluctuations in tension during contraction of single muscle fibers. Biophys. J. 20:315-334.
7. Iwazumi, T. 1987. High-speed ultrasensitive instrumentation for myofibril mechanics measurements. Am. J. Physiol. 252:C253-C262.
8. Ishijima, A., T. Doi, K. Sakurada, and T. Yanagida. 1991. Sub-piconewton force fluctuations of actomyosin in vitro. Nature. 352: 301-306.
9. Molloy, J. E., J. E. Burns, J. Kendrick-Jones, R. T. Tregear, and D. C. S. White. 1995. Movement and force produced by a single myosin head. Nature. 378:209-212.
10. Colquhoun, D., and A. G. Hawkes. 1995. The principles of the stochastic interpretation of ion-channel mechanisms. In Single-Channel Recording, 2nd ed. B. Sakmann and E. Neher, editors. Plenum Publishing, New York. 397-482.
[Author Affiliation]
Justin E. Molloy
Division of Physical Biochemistry, Medical Research Council National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom
[Author Affiliation]
Submitted April 5, 2005, and accepted for publication April 13, 2005.
Address reprint requests to Justin E. Molloy, Division of Physical Biochemistry, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. E-mail: jmolloy@nimr.mrc.ac.uk.
� 2005 by the Biophysical Society
0006-3495/05/07/1/02 $2.00
Cyprus is seeking U.S. support in talks on reunification of the ethnically divided island.
Cypriot Foreign Minister Markos Kyprianou was discussing the newly restarted reunification talks Friday with U.S. officials, including Deputy U.S. Secretary of State John Negroponte.
Cyprus was split into an internationally recognized Greek Cypriot south and a breakaway Turkish Cypriot north in 1974. Turkey had invaded in response to a coup by people who wanted to unite the island with Greece.
Greek and Turkish Cypriot leaders agreed last month to restart talks. The momentum followed the election of Cypriot President Dimitris Christofias in February.
Keegan Herring rushed for 116 yards, including a 71-yard touchdown, and Rudy Carpenter threw for 200 yards and another score Saturday to lead No. 9 Arizona State to a 24-20 victory over UCLA.
The Sun Devils (9-1, 6-1 Pac-10) remained in the thick of the conference race and kept their hopes alive for a spot in a BCS bowl.
The injury-riddled Bruins (5-5, 4-3) lost for the fourth time in five games.
Herring and Carpenter gave Arizona State just enough to overcome the Bruins, who kept the game close despite using their fourth quarterback of the year and starting their third-string running back.
Carpenter went 16-of-31, including a 9-yard touchdown pass to Kyle Williams. Chris McGaha had nine catches for 123 yards.
Osaar Rasshan, who had been converted to wide receiver then switched back to quarterback last month after injuries sidelined the Bruins' first two quarterbacks, went 14-of-27 for 181 yards, with one interception. He also ran for 41 yards on 13 carries.
The Sun Devils fell behind 10-0 early, trailed 13-10 at halftime, then went ahead to stay on Dimitri Nance's 11-yard touchdown run five minutes into the third quarter.
The teams traded touchdowns during a 13-second span of the third quarter. Herring reeled off his long scoring run to put Arizona State up 24-13, but Matthew Slater returned the ensuing kickoff 89 yards to cut the deficit to four points.
That ended the scoring.
The Sun Devils were coming off their lone loss, a 35-23 defeat by No. 3 Oregon in Eugene. The Bruins' recent losses include a 20-6 defeat by Notre Dame, still the Irish's only win of the year.
Both long scoring runs by the Sun Devils and Bruins were spectacular. Herring seemed trapped behind the line when he darted around right end, cut to the center, then sprinted down the left sideline.
On Slater's UCLA-record setting third kickoff return for a score this season, the Bruins' blockers sealed off the center of the field and he broke up through untouched, headed for the right side and outraced the final defender.
Down by six after Kai Forbath kicked a 42-yard field goal 23 seconds before halftime, the Sun Devils narrowed the gap to 13-10 by turning a mistake by UCLA defensive end Bruce Davis into a 53-yard field goal by Thomas Weber.
Carpenter threw a pass from his own 49-yard line that was knocked down in the end zone as time ran out in the second quarter. But Davis was called for roughing the passer, the penalty moved the ball to the UCLA 36, and ASU given an automatic first down with no time on the clock.
Weber, a redshirt freshman, then kicked his career-long 53 yarder. He has made 19 of 20 this season.
Forbath kicked his second field goal of the afternoon after a Bruins' drive stalled at the ASU 25, seemingly sending UCLA into halftime with a 13-7 lead. But Carpenter completed a 12-yard pass to McGaha to the 49, then two plays later, Davis drew the costly penalty.
After Forbath kicked a 28-yarder to cap a 51-yard drive on the Bruins' second possession, the Bruins held the Sun Devils to a three-and-out, and Terrence Austin returned Thomas Weber's 38-yard punt 68 yards to the ASU 1.
Chane Moline was stopped for a 1-yard loss on first down, but Craig Sheppard bulled over from the 2 on second down to give UCLA a 10-0 lead. Moline, the third-stringer who started at tailback, carried six times for 7 yards. Sheppard, a walk-on, gained 56 on 12 carries.
Defenses dominated the first half, with Arizona State held to 88 yards, just 12 rushing, and UCLA held to 114 yards, only 29 through the air.
The Sun Devils finished with 352 yards, and the Bruins had 300.
Slater's kickoff return was his third for a touchdown this year, setting both a season and career record for UCLA. The three in a season also tied the Pac-10 record set by Southern California's Anthony Davis in 1974.
The number of U.S. homeowners swept up in the housing crisis rose further last month, with foreclosure filings up nearly 50 percent compared with a year earlier, a foreclosure listing company said Friday.
Across the United States, 261,255 homes received at least one foreclosure-related filing in May, up 48 percent from 176,137 in the same month last year and up 7 percent from April, RealtyTrac Inc. said.
One in every 483 U.S. households received a foreclosure filing in May, the highest number since RealtyTrac started the report in 2005 and the second-straight monthly record.
Foreclosure filings increased from a year earlier in all but 10 states. Nevada, California, Arizona, Florida and Michigan had the highest statewide foreclosure rates.
Metropolitan areas in California and Florida accounted for nine of the top 10 areas with the highest rate of foreclosure. That list was led by Stockton, California and the Cape Coral-Fort Myers area in Florida.
Irvine, California-based RealtyTrac monitors default notices, auction sale notices and bank repossessions. Nearly 74,000 properties were repossessed by lenders nationwide in May, while more than 58,000 received default notices, the company said.
In Nevada, one in every 118 households received a foreclosure-related notice last month, more than four times the national rate. In California, one in every 183 households faced foreclosure.
The combination of weak housing sales, falling home values, tighter mortgage lending criteria and a slowing U.S. economy has left financially strapped homeowners with few options to avoid foreclosure. Many can't find buyers or owe more than their home is worth and can't get refinanced into an affordable loan.
Making matters worse, mortgage rates have been rising, reflecting increased concerns about what the Federal Reserve might do to battle inflation. Freddie Mac, the mortgage company, reported Thursday that 30-year fixed-rate mortgages averaged 6.32 percent this week, the highest level in nearly eight months and up sharply from 6.09 percent last week.
Efforts by government and the mortgage industry to stem the tide of foreclosures aren't keeping up with the rising number of troubled homeowners, and critics say a Bush administration-backed mortgage industry coalition, dubbed Hope Now, is falling far short.
Rick Sharga, RealtyTrac's vice president of marketing, said foreclosures are unlikely to peak until sometime this fall, as more loans made to borrowers with poor credit records reset at higher levels. "I don't think we've seen the high point," he said.
About 50 to 60 percent of borrowers who receive foreclosure filings are likely to lose their homes, Sharga said. The rest are likely to be able to sell or refinance.
A new government report released Wednesday found that among mortgages held by Bank of America, Citigroup Inc. and seven other large banks, foreclosures climbed to 1.23 percent of all loans in March from 0.9 percent in October.
As foreclosed properties pile up, they add to the inventory of homes on the market and drag down home prices. The trend is most dramatic in many parts of California, Florida, Nevada and Arizona, where prices skyrocketed during the housing boom and are now falling precipitously.
Sales of foreclosures, vacant new homes and other distressed properties now dominate some markets, causing grief for individual homeowners who need to sell for other reasons, like a job in a new city.
Nationwide, one out of every four sales between January and March was a distressed sale, and that figure jumps to more than 50 percent in the hardest-hit areas like Las Vegas, Detroit and distant suburbs of Los Angeles, according to Moody's Economy.com.
In some neighborhoods, lenders are slashing prices dramatically to rid themselves of an unprecedented number of foreclosed properties, sparking bidding wars and multiple offers.
While that's a positive for the real estate market, buyers in other parts of the country are still holding back. "I think a lot of people are waiting to see if we really have hit the bottom," Sharga said.
Lehman Brothers economist Michelle Meyer said in a report Thursday that U.S. home sales are likely to hit bottom at the end of this summer, but said a recovery in sales is likely to be "feeble." Home prices, she wrote, are still expected to fall another 10 percent by the end of 2009.
___
On the Net:
RealtyTrac Inc.: http://www.realtytrac.com
I'm really sick of this tit-for-tat regarding 527s. You know what 527s are by now. You should. As a result of the McCain-Feingold campaign-finance bill signed into law by President George W. Bush on March 27, 2002, we now have the same president calling for a ban on these groups he helped create.
I think it's best if we use the president's own words from a speech he gave immediately after signing the bill into a law.
"This law will raise the decades-old limits on giving imposed on individuals who wish to support the candidate of their choice, thereby advancing my stated principle that election reform should strengthen the role of individual citizens in the political process."
"I believe individual freedom to participate in elections should be expanded, not diminished; and when individual freedoms are restricted, questions arise under the First Amendment."
"I also have reservations about the constitutionality of the broad ban on issue advertising, which restrains the speech of a wide variety of groups on issues of public import in the months closest to an election. I expect that the courts will resolve these legitimate legal questions as appropriate under the law."
I have to say I agree with the president's comments two years ago. His regime's solution of banning all 527s reeks of censorship and may be unconstitutional. The solution, however, is simple. Any advertisement aired over the public airwaves (owned by the public and managed by the FCC) would have to be approved by a bipartisan committee with representatives from all political parties. Only unanimously approved ads could run. That'd keep it clean.
Article copyright Bar Bar Inc.
PEELING ONIONS VOLUME 1 by Lezley Davidson, 100 pgs First Choke Books #2 460 Tennyson Place, Victoria BC, V8Z 6S8,firstchokebooks.ca
Part art journal, part autobio strip, Peeling Onions is the first in a collection of Lezley Davidson's regularly updated webcomics, originally serialized at peeling-onions, com. Like any webcomic's first volume, you have to give it some time to find its footing. With this in mind, we are treated to a fairly bland beginning as Davidson gets into her groove. Early strips about the day-to-day of working retail at your own store are sometimes amusing, but impersonal and generally unremarkable, the filler of the webcomics world. As volume 1 goes on, …
Eastman Kodak Co. said Thursday it lost $189 million in the second quarter, its third consecutive quarterly deficit, as the global economic downturn hurt sales of digital cameras and other photography products.
The photography pioneer lost the equivalent of 70 cents a share in the April-June period, compared with profit of $495 million, or $1.62 a share, in the same quarter a year earlier.
The latest quarter included one-time charges of $75 million, or 28 cents per share, mainly related to restructuring and taxes. Excluding one-time items, Kodak lost $116 million, or 42 cents per share, in the latest period.
Sales plunged 29 percent to $1.77 billion.
Analysts polled by Thomson Reuters expected Kodak to post a loss of 37 cents per share on sales of $1.83 billion.
Revenue from digital businesses dropped 28 percent to $1.17 billion and traditional film-based revenue fell 30 percent to $593 million.
Kodak said its results reflected investments in new higher-margin digital cameras, video cameras and other consumer electronic products. Those moves, it said, will help lead to improved cash and earnings in the second half of 2009.
"We have every expectation that our cash flow pattern this year will mirror the pattern of previous years, with a sizable increase in cash generation in the second half," CEO Antonio Perez said in a statement.
Kodak spent $3.4 billion from 2004 through 2007 converting the bulk of its 129-year-old business from high-margin film to more competitive electronic technology. It is in the midst of cutting 3,500 to 4,500 jobs, which could reduce its work force to a 1930s-era low of 19,900 from a 1988 peak of 145,300.
Its consumer digital imaging division absorbed a $99 million operating loss in the quarter compared with a year-ago loss of $49 million as sales fell 33 percent to $503 million.
Hurt by a softer commercial printing market, graphic communications posted a $28 million operating loss, compared with a $13 million profit a year earlier, as sales fell 24 percent to $670 million.
The film, photofinishing and entertainment unit had an operating profit of $51 million, down from $54 million, as sales fell 30 percent to $593 million.
Rochester, New York-based Kodak is bracing for an estimated 12 percent to 18 percent slump in sales this year because of the recession. Through 2012, it expects revenue to rise 4 percent a year on average, driven by an 8 percent to 10 percent increase in digital sales.
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On the Net:
Eastman Kodak moves to 2nd-quarter lossEastman Kodak Co. said Thursday it lost $189 million in the second quarter, its third consecutive quarterly deficit, as the global economic downturn hurt sales of digital cameras and other photography products.
The photography pioneer lost the equivalent of 70 cents a share in the April-June period, compared with profit of $495 million, or $1.62 a share, in the same quarter a year earlier.
The latest quarter included one-time charges of $75 million, or 28 cents per share, mainly related to restructuring and taxes. Excluding one-time items, Kodak lost $116 million, or 42 cents per share, in the latest period.
Sales plunged 29 percent to $1.77 billion.
Analysts polled by Thomson Reuters expected Kodak to post a loss of 37 cents per share on sales of $1.83 billion.
Revenue from digital businesses dropped 28 percent to $1.17 billion and traditional film-based revenue fell 30 percent to $593 million.
Kodak said its results reflected investments in new higher-margin digital cameras, video cameras and other consumer electronic products. Those moves, it said, will help lead to improved cash and earnings in the second half of 2009.
"We have every expectation that our cash flow pattern this year will mirror the pattern of previous years, with a sizable increase in cash generation in the second half," CEO Antonio Perez said in a statement.
Kodak spent $3.4 billion from 2004 through 2007 converting the bulk of its 129-year-old business from high-margin film to more competitive electronic technology. It is in the midst of cutting 3,500 to 4,500 jobs, which could reduce its work force to a 1930s-era low of 19,900 from a 1988 peak of 145,300.
Its consumer digital imaging division absorbed a $99 million operating loss in the quarter compared with a year-ago loss of $49 million as sales fell 33 percent to $503 million.
Hurt by a softer commercial printing market, graphic communications posted a $28 million operating loss, compared with a $13 million profit a year earlier, as sales fell 24 percent to $670 million.
The film, photofinishing and entertainment unit had an operating profit of $51 million, down from $54 million, as sales fell 30 percent to $593 million.
Rochester, New York-based Kodak is bracing for an estimated 12 percent to 18 percent slump in sales this year because of the recession. Through 2012, it expects revenue to rise 4 percent a year on average, driven by an 8 percent to 10 percent increase in digital sales.
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On the Net:
