I was there last week and I got a dance from a beautiful young lady, she was very nice but after we were done i noticed the manager dave approach the young lady and the way he was talking you wouldve thought he was a pimp. Besides Raven, the other best thing in the club was the DJ. Then after she was done, they lined up onstage again and it was back to the same ol same ol. It wasnt until we saw Raven who came sliding down the main pole and did a wonderful set that we got excited. The girls were beautiful, some knew how to actually use the pole, others would just stand around an move their hips, and the only conversation with the girls was them coming up ask if we wanted a lapdance and that was it. One of the girls was messing with her shoe for half of one of her songs and then snapped at us for sitting at the "tip bar" and not tipping her, but we were waiting for her to finish with her shoe. But we were there for 2 hours and saw only 4 girls dance, the rest of the time was birthday lapdances and then they would line up and do 3 for 1 lapdances. It was slow at the beginning of the night, so I dont blame the girls for not putting their all into their dancing. I know the club can not serve alcohol so they have a wide selection at the juice bar which was nice. Took my husband there for his birthday and my first strip club experience. I dont recommend this place to anyone not even my worst enemy or Hitler himself. The staff is bad, the managers dont actually do their job and after talking to the girls, you can figure out who the dirty ones are, the ones who give you the "happy ending". I dont even know how this place remains open. You can tell the managers dont care about them or anything else as the carpet is bunched up in a few places making it easy for the girls and customers alike to trip. They put up with bad customers & bad management who rip them off and disrespect them (I saw the way they talk down to the girls). The girls walk around with a chip on their shoulders and I cant blame them. Clearly theres an issue that doesnt seem to get addressed. They also dont regulate the floor when no one seems to be getting dances. The "tip rail" gets overcrowded with people that dont tip and management does nothing to fix the issue. The bartender ignores you and treats you like youre an inconvience when you come up for a refill or change and wont end his conversation or put his phone down to help you. Walking in, youre greeted by a door guy with a poor attitude and a high cover and drink charge. Police said the shootout suspects left the scene, and no arrests were made.Management and staff is honestly the worst. "If you’re looking at that footage, that does not appear to be people unhoused, that appears to be people around cars they have driven there or ridden in," O’Connell said.įreddie said something needs to be done so residents can feel safe again. they responded to both weekend incidents within 5 minutes from when they were dispatched. Ironically, our club is the only safe haven for visitors in the area.” The Police Department doesn't respond for hours when called and unaffiliated parking lots are left to decay into nightly demonstrations of violence, drugs, and misdeeds. The homeless population and well-known crack houses up the street are destroying this neighborhood. Unfortunately, due to the negligence of the Police Department and neighboring property owners (in particular, one very negligent parking lot owner), the Church Street area has become unsafe at times. “This altercation was on a property completely unaffiliated with Deja Vu and among people who have never been inside our building. Ryan Carlson, the CEO of Déjà Vu Showgirls Nashville issued a statement: "This has been a hotspot, and we need to focus on it like it’s a hotspot," O’Connell said, "The likelier it is being neglected by authorities across multiple departments, the likelier it is that activity starts to occur there on a regular basis." People who live or work nearby reached out to Metro Council Member Freddie O'Connell as they're worried. The unconscious man was rushed to the hospital after medics did chest compressions according to the Nashville Fire spokesperson. Then the next day, a police incident report said a man was found by strip club staff unconscious in a bathroom stall with possible drugs by him. Shell casings are scattered around the parking lot, and a vehicle was hit multiple times with bullets. there was a shootout in a parking lot that's leased by several businesses at 15th Avenue North and Church Street. (WTVF) - Some people are concerned about crime after a shootout outside Déjà Vu Showgirls Nashville, but the CEO said it’s not their fault.Įarly Saturday around 2:30a.m.
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The I-Team reached out to several office store chains to see how they safeguard consumers’ private information. “Our handheld phones, our smartphones, are not just phones - they are little computers. Our fax machines and our printers can be ports into our servers,” Velasquez said. “We really have to shift our thinking that these wonderful devices are in a bubble. Her advice: be your own privacy advocate, which means being more aware of where you plug in or print. “About five years ago, several fraud rings were busted buying recycled copy machines where all the images were saved in the hard drive, and probably the more concerning was police departments had recycled their copy machines and all the photocopies and police reports were in the hands of the criminal element,” said Eva Velasquez, who runs the Identity Theft Resource Center. “There is a risk if you leave it inside,” the business owner said.Īnd if the printer at your corner copy store or your office is sold to a new owner, your information could go with it. “All the details that you print or scan, all the information is there,” said one local copy machine store owner.ĮZ Office Machines in Los Angeles buys used copiers and pulls out the old drive before reselling them. Inside every modern copier lives a hard drive that automatically stores every image that’s printed. “I told them – ‘I’m sorry’ is fine, but that doesn’t give me peace of mind,” Deborah said.ĭrive mix-ups aren’t the only risk you could encounter at your local office store. Employees told her they found it in a store safe, but she believes some of her documents were missing.Īfter the company was contacted by the I-Team, a Staples spokeswoman said “we are not aware of any protected personal information being compromised,” but, “we have improved our labeling procedures so this doesn’t happen again.” “Oh, I feel so violated,” the woman said.ĭeborah said it took Staples 24 hours to track down her missing drive. “I noticed other people’s files, someone’s tax returns, invitations, car dealership and some were in Spanish,” she said.ĭeborah used the tax returns to track down the drive’s owner, an Orange County businesswoman who asked NBC4 to not be publicly identified because she is worried the breach might panic her customers. ‘Out of Nowhere': Cessna Pilot Makes Emergency Landing on Ventura Beach Meanwhile, just like Buckner, veteran actress Dawn Wells is also a celebrated film and television actress. However, she is still considered one of the most successful actresses in the industry because of her iconic roles. The actress also appeared in the 2018 television series "Behind Closed Doors." She was simply herself in the documentary-style series.īuckner had a short-lived career in acting. In 1981, Buckner starred in the hit slasher film "Deadly Blessing." She has taken part in various documentaries since then. However, Miss Ohio Laurel Lea Schaefer went on to win the title. In 1972, Buckner became a top ten finalist in the Miss America pageant. In 1971, she was crowned Miss Washington. It also became the highest-earning musical film ever.īefore becoming a successful actress, Buckner was actively involved in beauty pageants. The flick has grossed a domestic total of $188,755,690 and a worldwide total of $394,955,690 as of 2017. Jami Donnelly, Didi Conn Barry Pearl, Randall Kreisser, Susan Buckner, Edd Byrnes, Lorenzo Lames, Michael Badalucco, and Len Delessio at Hilton Parsippany on Apin Parsippany, New Jersey. But that was more than any other celebrated movies that year.īefore becoming a successful actress, Buckner was actively involved in beauty pageants. In 1978, "Grease" grossed only under $160 million domestically. It was written by Bronte Woodard and directed by Randal Kleiser. The movie was based on the 1971 musical of the same name by Jim Jacobs and Warren Casey. She then appeared in the 1977 TV series "The Hardy Boys/Nancy Drew Mysteries."īuckner gained worldwide popularity when she starred as high school cheerleader Patty Simcox in the musical romantic comedy film "Grease." She co-starred with other veteran actors - Olivia Newton-John and John Travolta. Susan Buckner, who was born in January 1953, received her big break as a dancer on "The Dean Martin Show" in 1971. Susan Buckner shot to fame when she was picked to star as Patty Simcox in the film "Grease." Much has happened to the actress since the movie debuted in 1978. Susan Buckner at Hilton Parsippany on Apin Parsippany, New Jersey. My shopping list was simple: Bacon and Tortilla Chips. I was a little slow at getting to it today and I kept pushing off going to the store until later in the day and when I went, I just went up the street to the Dollar General (a noted purveyor of fine meats). I don’t have to keep up with the Joneses (who are probably making bacon-wrapped waffles in bacon syrup served with bacon beer). I need to stick to the basics and just celebrate. You have to pace yourself over this journey. I’ll leave that to others while I prepare for the 200 plus days left of this quest. I just don’t have to put on the biggest celebration for it. Sure I am going to celebrate bacon and I will do so happily. Then I realize that this is a long quest, not a one day thing. You start seeing it pop up on social media and trending and you start questioning how am I going to make my mark out there? I don’t want to let down my fans. When you celebrate something like bacon, you feel you have to come out strong and up your game. It’s easier to celebrate a day like Pepper Pot Day because not too many people know what it is. I always have a tough time on holidays that are bigger and more widely recognized than my usual celebrations. The world knows that men love bacon and they are cashing in on it. If you have ever checked the “Man” section at a gift store (an awful section of any store that reduces my gender to a group of bourbon drinking, sports watching, bacon eating buffoons), there’s always an area devoted to the love the of bacon. The world knows this and cheers for bacon at every chance it gets. It’s pretty versatile and it will always make whatever you are eating better (ok, maybe not if you were eating Jell-O or something like that). You can wrap anything in bacon and make it better. Just a plain old Townhouse cracker with a little bit of cheese. But if you really look at what I served – it was essentially a cracker. On Christmas, I served bacon covered crackers that were a big hit. It makes anything it attaches to more tasty as well. Salty, fatty, crispy and oh so flavorful. It’s everything you want in a meat product. Histera is developed and published by StickyLock Games.There are few foods in the world that are celebrated more than bacon, and rightfully so. As a community member, you will be able to see how the game goes from concept to final product, make suggestions for improvements, vote on new features, join public playtests and much more! Instead of just throwing a game out there, StickyLock Games decided to get the community involved throughout the development process. “We are working hard to bring it to Steam for early access by the end of this year.” “By using the latest Unity DOTS and Netcode we can bring some unique features to this game,” said lead developer Mitchel Alberts. StickyLock’s team of 40+ creative artists and developers have been working on the new title for the last 2 years. The game will also include several unique never-before-seen features and competitive e-sports style gameplay including an amazing level-editor option for all players. Experience vastly different surroundings, outfits and weapons that will force you to change tactics and resources. It is possible for players to run around in high-tech armor fighting a rebel uprising, or stomp around in primitive clothes with improvised weapons and fighting a rival tribe. Histera is a multiplayer first-person shooter that takes place in multiple simulated eras and moments in history as well as in the future. It hadn't been designed with the idea that someone would hold a ruler along with moving the quilt, but that's what I did. You just need to match the shank type of your machine to the foot.Ī ruler foot made to be used on the Janome 1600P when mounted on a frame system is what got me started with rulers. But now there are several options for ruler feet, both made by machine makers and after market feet, usually made by ruler companies for a wide variety of machines. Using rulers on a stationary machine when free motion quilting is still a bit of an out-of-the-box technique and until about two years ago, you may have had to try some out-of-the-box thinking about finding a ruler foot. When using a ruler foot, always lower the foot before lowering the needle. Many quilters tend to bring up the bobbin thread with the foot in the up position and then lowering it for sewing. The very simple habit of lowering the ruler foot into the stitching position before lowering the needle and not raising the foot when the needle is down solves this issue. This also has potential to damage your machine. This means that the clamp and bar that holds the needle in position can run into the top of the foot if the needle is lowered while the foot is in the raised position. A machine 'out of time' will have trouble forming stitches.Īdditionally, the high edge of a ruler foot reduces the clearance between the needle bar and the foot on a domestic sewing machine. Without the sturdy, high edge around the needle, you run the risk of having a ruler slip over or under the foot and breaking the needle and possibly throwing the machine out of timing. Using a "ruler foot" is highly important with this technique. These machines usually have a ruler foot available and the content I provide here is applicable to these machines. Sit down long arm systems have become popular in the last few years and these machines use the same "move the quilt" method of quilting as those who quilt on a sewing machine. It took some practice, but is totally doable! After some research, other feet were found (and some created by others) to use with this technique. I first experimented with a Janome ruler foot that was intended for use with a machine in a frame system, instead moving the ruler and quilt along the foot as a unit. See the ruler feet by clicking this link.Įvery single bit of this quilt in progress was quilted with rulers. My recommendations and descriptions are detailed and informative, service is fast, and shipping is reasonable. It can also help more experienced quilters by giving more structure to a quilting design.Įdited Septemto add: I am excited to let you know that Amy's Quilting Adventures now carries a wide variety of quilting rulers and the feet needed to use them safely. Using rulers to quilt can be a little like training wheels for a new free motion quilter, helping to form designs. I didn't create ruler work, rather I found a ruler foot and adapted what long arm quilters were doing with rulers, which allows the creation of straight lines and smooth curves. Using rulers to guide free motion quilting on a domestic machine is my signature technique. Decatur “repeatedly raked his opponent, and, without losing a man, finally forced her surrender with 50 of her crew of 84 killed or wounded.” On October 31, 1803, the PHILIDELPHIA, a 36-gun frigate, ran aground on an uncharted shoal while pursuing a small craft in Tripoli Harbor. Tiny ENTERPRISE handed the Barbary pirates their first defeat against the better-armed ship, TRIPOLI. Stephen Decatur’s command, the third ENTERPRISE (a small, 12-gun clipper schooner) was deployed to the Mediterranean in 1801 as the smallest member of a four-ship squadron assigned to protect commerce from the Barbary States of Morocco, Algiers, Tunis, and Tripoli. The Stephen Decatur Leadership Award was presented to an outstanding junior officer. Holloway participated in the Washington, D.C., program for ENTERPRISE leadership award winners every year until his health prevented it. Navy encompassed combat in three wars and culminated in service as the 20 th chief of naval operations from 1974-1978. His 39 years of service on active duty in the U.S. Holloway III was the third commanding officer of the USS ENTERPRISE (CVN-65), serving from July 1965 through July 1967. Holloway III Leadership Award was presented to an outstanding senior officer. That action is consistent with the heroic service of CVN-65 and the previous seven ships to carry the ENTERPRISE name – all affectionately known by the nickname “Big E.” When the USS ENTERPRISE Leadership Awards for outstanding leaders on the USS ENTERPRISE were established, each award was named after an exceptional leader who served America on an ENTERPRISE-named ship: Winnefeld immediately turned the ship around to be in position to launch the first air attacks against Al-Qaeda off the coast of Afghanistan days later. As the ship was returning home from a six-month deployment to the Persian Gulf on September 11, 2001, news broke of the attacks on the World Trade Center in New York, the Pentagon in Washington, D.C., and the hijacking of United Airlines Flight 93, which crashed over Pennsylvania. “Sandy” Winnefeld (USN, RET), who later served in many key national defense leadership positions, including vice chairman of the joint chiefs of staff. The Stennis Center’s partnership with the USS ENTERPRISE began in 2000 when the ship was under the command of Adm. The ninth ship to carry the name, the USS ENTERPRISE (CVN-80), is scheduled for completion in 2027. The eighth ship to bear the name, the USS ENTERPRISE (CVN-65) aircraft carrier, was a part of the Stennis Center’s leadership awards program until her decommissioning in 2017. National Archives or DVIDS.ENTERPRISE is a storied name in American military history, proudly borne by ships in service of the United States since the Revolutionary War. This website is developed as a part of the world's largest public domain archive,, and not developed or endorsed by the U.S. law and are therefore in the public domain. National Archives and DVIDS is "a work prepared by an officer or employee" of the federal government "as part of that person's official duties." In general, under section 105 of the Copyright Act, such works are not entitled to domestic copyright protection under U.S. All of these materials are preserved because they are important to the workings of Government, have long-term research worth, or provide information of value to citizens.ĭisclaimer: A work of the U.S. The Defense Visual Information Distribution Service provides a connection between world media and the American military personnel serving at home and abroad. There are approximately 10 billion pages of textual records 12 million maps, charts, and architectural and engineering drawings 25 million still photographs and graphics 24 million aerial photographs 300,000 reels of motion picture film 400,000 video and sound recordings and 133 terabytes of electronic data. NARA keeps those Federal records that are judged to have continuing value-about 2 to 5 percent of those generated in any given year. National Archives and Records Administration (NARA) was established in 1934 by President Franklin Roosevelt. National Archives and Defense Visual Information Distribution Service. The objects in this collection are from The U.S. Said another way, we need to preserve the written history of our hobby while the original magazines are available to us. We are pleased to display nearly three dozen replicated models from the history of the hobby.Īdditionally, we have created the Scan the Past program which focuses on the goal of digitalizing significant model car magazines and related publications that have chronicled the history of our hobby. This program is an essential "core" effort because there needs to be a way of acknowledging, and understanding, the history of the technological development of model car construction since the hobby first emerged. In 2018, we reached out to the Fisher Body Craftsmen Guild craftsmen and now host a major exhibition of the work of those craftsmen from so long ago.Īs part of the effort to chronicle and celebrate the history of our hobby, we have created the Hot 150/Clone the Past program with its accompanying display case that identifies the most influential models in the history of our hobby, and then to either acquire/restore the original model, or replicate and present the same in a chronological display. We have a large library, display cabinets dedicated to model car topics of all descriptions, and we collect memorabilia from contests and displays from around the world. The Museum has display cases filled with the models of the famous and the obscure alike, each divided into topical displays: street rod, customs, replica stock and competition. Instead, our goal has been to collect as much information and as many artifacts about services and supplies offered to hobbyists, and to focus on those individuals who have expressed their craftsmanship and creativity by constructing scale miniature automobiles. The Museum isn't about collecting kits, though we have a modest collection of rare items. Our initial effort has been to gather and chronicle the history of the model car hobby from the perspective of the builder. The mission of the Museum is multifaceted. The International Model Car Builders' Museum was founded with a simple goal: Preserve the memories of the scale vehicle hobby for all hobbyists - old and new – and promote and support contemporary contests and displays which encourage current builders. They require more space to carry them safely. The mover will place a higher cost on such items because Items such as artwork, musical instruments, and decorative fixtures are prone to damage and demand extra care. The fragility of your itemsįragile and delicate items may demand a higher price because they need special care. Therefore, if you can save client time by ensuring everything is packed and ready to go, you can reduce The longer a mover stays loading or unloading your belongings, the more it will cost. Labor time influences the moving quotes and cost of your move. Longer to load and unload, which is why the cost is high. If you have more things to move, you will pay more in expenses because it requires a bigger truck. Cargo weight is a vital factor in influencing the cost. The weight of your belongings also determines the quotes and prices. Hence, bigger size will demand higher costs to move the home since quotesĪre based on the weight and volume of your belongings. A minimalist living in a studio department will have fewer belongings than someone living in a fully-furnished four-bedroom home. The size of your home can affect your moving costs. These estimates are based on a three-bedroom move of up to 7,500 pounds. In the United States, the average cost of a local move is $1,250, while a long-distance move Expect higher costs if you are moving a long distance to your new destination. The distance of your move between the old and new home influences the final cost. Factors Influencing the Moving Cost Distance The hourly rates will vary between $25 and $85 per hour per mover,ĭepending on your location and the time you move. The formula for calculating the cost of your move will be (hourly rate) x (number of movers) x (number of hours to complete the move) + travel fee and charges for additional services. A time feeĪnd any requested extra services will be added to the final price. Most local movers charge hourly by tallying up the time between the origin and final destination, including the time to load and unload your belongings. If you are moving a short distance, time will be a crucial factor in estimating the cost. In such cases,Ī moving inventory will get a rough idea of the cost depending on the weight and distance. When moving a long distance, the costs will depend on your shipment's weight and the actual distance to the destination. The actual moving costs and quotes depend on several factors, with weight and distance playing a significant role. You plan your finances and select an affordable moving company. Here is a guide on getting a moving estimate to help Moving to the next block or cross country, you need to get a professional moving company to make a move for you. However, you can struggle to get an accurate company that provides a true sense of what you need. In most cases, you won't have a problem finding moving companies with thousands available to choose from. Free Moving Quotes: Everything You Need to KnowĪ free moving quote from professional companies can give you a starting point for making cost comparisons across various moving companies. We accept the magic circle of games as being completely separate from real life. In games we see the magic circle as a structure of formalized rules, norms, and excepted behaviors. In these places we’re called to act and perform differently according to a new set of rules. The magic circle can be observed in court houses, religious spaces, and even special events like weddings. Interestingly, these rules are also not limited to only games. It’s the idea that this location becomes a dimension in space and time where the imagined becomes real and where new rules apply for new circumstances. That’s because the magic circle represents this boundary. For gamers this could be gathering around a table with friends online with their guild or even on the baseball diamond or football field. When we enter the magic circle we undoubtedly enter a separate, special, and magical place. We create a time and place where the “world’s rules” are now the “game’s rules.” This means that playing within the magic circle creates a temporary and provisional dimension. They are created because game rules dictate what we can and cannot do as well as what is supported and endorsed by other players. They are created by players, with players, and within the game. In games, we represent battles via avatars on a game board. In games, our characters progress up skill trees. In games our health become quantified into “hit points” (HP). Game actions taken in the magic circle have a different meaning and impact inside the game. The most important thing to consider with games in the magic circle is that actions within the circle create a different kind of meaning for players. Other times the magic circle exists only to provide a boundary between the real and imagined worlds. Sometimes there are “correct” ways to play in the magic circle. Sometimes it’s cooperative, competitive, or both. The magic circle is the place that we enter when we begin playing games. The “Magic Circle” is when and where games are being played and new meaning is generated. So what is the “magic circle” really? The idea and concept of the magic circle is a relatively straight forward and simple idea. Finally, this article closes on using the magic circle in games-based learning as well as some criticisms around its use. Players’ rules and roles within the circle as well as their return to “reality” are covered in this article. Characteristics and locations of the magic circle will be discussed as well as how players cross into the magic circle. This article will answer the question “What is the magic circle?” In addition, it’ll address the magic circle as the “separate and special place” in gaming. That type of escape is into the “magic circle” of games. The magic circle is a place where different rules that govern our existence, actions, and consequences applyīut what is this “magic circle?” Where does it exist? Are players always in the magic circle? What do they do within the circle? How do players create meaning inside the magic circle? Most importantly: how can we use the magic circle in games-based learning? We escape to a faraway place that is as different and distinct from the place that we call reality. One of the reasons why we play games is often to escape. searchs always games, covers, posters, manuals or solutions of Commodore 64 software for the next update.We may play games for various reasons. Together on this homepage you find now totally 7.162 programs for a C64. Following games are new on this homepage:ġ581 Toolkit, Atari Soft Collection, Boom (Prince Phase 101), Boulder Dash Junior 5, Boulder Dash Junior 6, Bundesliga Manager V1.1, Crapoid, Cross Bomber, Diamond Jones, Diamond Jones II, Dot Gobbler v3, Duell-Tris, Galaxian DX, Galaxian I, Galaxian II (Courbois Software), Galaxian of Xiabex, Galaxians, Luna - Xmas Edition, Main Frame, Newquarts, [[P1X3ILPushrv, Park Adventure, Petscii Lemmings, Phoenix Recovered, Pic-Man, Popeye (Atari Home Computers), Kaiser, Kaiser Englisch, Buck Rogers - Countdown to Doomsday, Champions of Krynn, Curse of the Azure Bonds, Death Knights of Krynn, Dot Gobbler v3, Duell-Tris, Erotika 1, Frank Brunos Boxing, Katabatia, Luna - Xmas Ediition, Magnetron (Broderbund), Moonwalker, Pool of Radiance and Project Firestart. All measured masses are summarized in Table 1. The two most prominent particle populations represent the empty (∼23,000 particles, MW = 3.85 MDa) and genome-filled rAAV6a particles (∼40,400 particles, MW = 4.63 MDa), with a relative mass difference of ∼800 kDa, due to the packaging of the expected genome. From 2D CD-MS, two prominent co-occurring distributions can be observed, but also two weaker particle populations, all with distinct masses. Cumulatively, the 2D CD-MS spectrum shown in Figure 2A shows the data for about ∼76,000 individual rAAV6a particles. By CD-MS, the obtained two-dimensional (2D) histogram and the corresponding mass histogram for an AAV6a serotype, expressed with the above-mentioned transgene, are shown in Figures 2A and 2B. Besides packing this genome, other possible genome variants that may be packed by the AAV are a self-complementary ssDNA (MW ∼2 × 800 kDa) and truncated forms of the genome as further depicted in Figure 1. The analyzed particles were expressed with a common expression cassette with an approximately 2.5-kb long genome (with a molecular weight of ∼800 kDa). The importance of using this optimized acquisition approach becomes most evident when aiming to analyze and quantify low-abundant encapsulated genome variants, as demonstrated in Figure 2. Results Optimizing the analysis of co-occurring AAV particles in CD-MS This new approach can contribute to the optimization of the bioprocessing of rAAVs, producing more particles containing the desired intact transgene. We show that the quantitative accuracy of the CD-MS method to distinguish filled from empty particles is in the range of 1%–3% (see Table S1). We introduce an improved acquisition method with better signal utilization that can accurately and reproducible detect rAAV particle populations as low as 2%, also identifying some less-abundant, likely scAAV, variants as off-target products in rAAV preparations. We determine the mass and abundance of various co-occurring rAAV particles in less than 30 min, using only 1 μL of sample at typical AAV working concentrations (2 × 10 13 vc/mL). Here we present an optimized workflow enabling the mass analysis of heterogeneous rAAV particles, whereby the mass resolving power and accuracy attainable allow us to determine and quantify genome integrity. Here, Orbitrap-based CD-MS is explored for quality control, as it is sensitive, facile, and can yield information on capsid/genome integrity. For monitoring this, AUC and EM are the industry standards, whereby the former can also serve as preparative method. This efficiency is sensitive to the presence of empty capsids and capsids filled with off-target genomes. Detailed characterization is important as only a small number of the infectious particles will transduce their genome. rAAV capsids show additionally a wide distribution of empty, partially filled, and filled capsids, requiring additional purification and monitoring. The initial number of other contaminants (DNA, side product, host cell proteins) can be several orders of magnitude higher than that of rAAV capsids and require extensive purification. These gene products are all encapsulated (5) and will yield a mixture of empty and (partly) filled particles. Other encapsulated off-target genomes could originate from self-complementary ssDNA dimeric variants (3) as well as truncated genomes (4). After transfection of (or infection with) the corresponding transgene (1) (if replicated properly) they will yield an encapsulated 800-kDa genome (2). Schematic overview of rAAV production in either HEK293 or SF9 host cells for a designed transgene of around 2.6 kb. The method quickly assesses the integrity and amount of genome packed AAV particles to support AAV bioprocessing and characterization of this rapidly emerging class of advanced drug therapies. A protocol is presented that allows the quantification of genome-packed AAV preparations in under half an hour, requiring only micro-liter quantities of typical AAV preparations with ∼10 13 viral capsids per milliliter. Here we show that Orbitrap-based charge-detection mass spectrometry allows the very sensitive quantification of all these different AAV bioprocessing products. Current methods to monitor genome packaging have limited sensitivity, a high demand on labor, and struggle to distinguish between packaging of the intended genome or unwanted side-products. Genome packaging is an essential step in the bioprocessing of AAVs and needs to be tightly monitored to ensure the proper delivery of transgenes and the production of effective drugs.
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