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Genetic Engineering & Biotechnology News | GENengnews.com | JULY 2016 | 29 microcarriers could settle, and the reactor was drained to 20% volume using the upper harvest line. The reactor was then re-fed with virus pro- duction media to full 2.0 L volume using a top feed line. The production media (DMEM w/glucose + 4 mM L-glutamine, 1% NEAA, and 1% sodium pyruvate) contained infuen- za virus (strain A/WS/33) at a multiplicity of infection (MOI) of 5 × 10 -4 pfu/cell. Agitation was then resumed at 90 rpm and the tempera- ture reset to 33°C (the optimal growth tem- perature for this viral strain). After 72 hours, the virus was harvested. Typical viral harvest titers were between 2.0 x 10 4 to 4.5 × 10 4 hemagglutination units (HAU)/mL. Large-Scale Cell Attachment The 3L protocol was scaled up for MDCK cell growth in a Mobius 50L bioreactor using Cytodex 1 microcarriers. Some adjustments for the larger scale were empirically deter- mined. Optimal cell attachment in the reac- tor was performed at 25 L working volume with an impeller speed of 70 rpm (~40 cm/s). An additional consideration was that the Mobius 50L employs an external pump to circulate the culture through the Sensor- Ready ® loop to perform inline sampling of pH and DO for feedback control. Dur- ing the inoculation step the SensorReady pump speed was set to 350 rpm to allow for cell attachment and feedback control of pH and DO. Once cells were observed to be fully attached, the SensorReady pump speed was increased to 700 rpm to prevent settling of microcarriers and cells during the growth phase. Large-Scale Cell Growth For cell growth in the 50L (25L working volume) bioreactor, agitation at 70 rpm was optimal for suspension of the microcarriers. Sparging in the 50L was performed using the microsparger with both air and oxygen, which kept foam accumulation to a mini- mum during the growth phase without the use of antifoam. Cells were grown for four days at 37°C to a density of 1.8 × 10 6 vc/mL. DO and pH control were the same as 3L. These methods produced very similar cell growth kinetics between the 3L and 50L scales (Figure 2). This study demonstrated that Mobius bioreactors are a suitable platform for scal- able growth of adherent cells and viral vac- cine production. The Mobius single-use bio- reactor line also includes 200L and 2000L sizes, and future work will extend these pro- tocols to these larger scales. Also underway are studies to establish methods for using the smaller bioreactors to seed the larger, avoiding the need to use large quantities of 2D fasks to generate the cell seed. Celebrating 20 Years of Innovation and Discovery 1996 TriLink BioTechnologies is founded in May and operates out of a home in San Diego, California for several months while buying ofce and lab equipment at local auctions. In August, the company moves into its frst commercial lab. By year-end, TriLink ships frst oligonucleotide products and hires frst employee. 1998 First customer-written paper citing TriLink products is published in the Journal of Immunology. 2002 Company continues to experience remarkable growth and expands to a 40,000 square foot building that still serves as the corporate headquarters. 2006 Proven pre-clinical oligo production leads to frst alliance partnership with large therapeutic oligo CMO, enabling customers to easily transition from R&D; to clinical trials. 2014 Receives ISO 9001 certifcation. Opens state-of-the art GMP manufacturing facility and expands into mRNA and oligonucleotide therapeutics. Secures contract for synthesis of Ebola vaccine with Battelle. Awarded patents for proprietary CleanTag™ technology. 1997 Obtains license to synthesize radioactive compounds. TriLink quickly becomes the industry leader and one of the only companies in the world to successfully manufacture radio-labeled oligos. 1999 Expands into nucleotide triphosphates. Cofounds Solulink, a company focused on unique bioconjugation chemistries. Outgrows frst lab and expands to Nancy Ridge site. First paper under TriLink corporate name is published. 2005 Receives frst of four SBIR grants for chemically modifed primers and NTPs for improved hot-start PCR, the basis of the CleanAmp™ product line. 2011 Leverages expertise with complicated nucleic acids and begins IVT production of long RNA and mRNA for use in pharmaceutical research and development. First patent for CleanAmp™ is granted. 2015 Publications citing TriLink products exceed 6,000. Adds gram scale mRNA production and expands mRNA purifcation oferings. 9955 Mesa Rim Road San Diego, CA 92121 800.863.6801 | info@trilinkbiotech.com | www.trilinkbiotech.com BIOPROCESSING Tutorial > VWR Acquires JM Separations VWR has acquired JM Separations, a supplier of single-use fuid handling and separation technology products for biotech customers. JM Separations, based in Tilburg, the Netherlands, pri- marily serves customers across Europe and supplies complete single-use bag systems and custom solutions for me- dia/bufer preparation. VWR maintains its headquarters in Radnor, PA, and serves the pharmaceutical, biotechnol- ogy, industrial, education, government, and healthcare industries . > Horizon Discovery, ProteoNic Ink Licensing Agreement Horizon Discovery Group has signed nonexclusive licensing agreement with ProteoNic to provide ProteoNic's CHOGUN expression vector to custom- ers alongside Horizon's HD-B BIOP3 GS Null CHO cell line as standard. A GS null cell line (when paired with the cor- rect vector) increases the stringency of selection and enables identifcation of clones expressing biotherapeutic prod- uct at commercially viable levels, with reduced timelines and without the need for toxic methionine sulphoxi- mine as a selection agent. > Lonza, bluebird bio Partner on Manufacturing Lonza and bluebird bio have entered into a strategic long-term commercial manufacturing agreement for the fu- ture commercial production of bluebird bio's drug products, Lenti-D™ and Len- tiGlobin™. As part of the deal, Lonza will complete suite design, construction, and validation in addition to process validation prior to commercial launch. For its part, bluebird bio brings its ex- pertise in developing gene therapies for severe genetic diseases and T cell– based immunotherapies for cancer. This agreement encompasses pro- duction suites within Lonza facilities that are currently under construc- tion and expected to provide for the clinical and commercial supply of viral vectors and virally modifed cell therapy products. > Pfzer Breaks Ground on $200M Manufacturing Facility Pfzer broke ground on a $200 million biologics clinical manufacturing facility in Andover, MA, a project that will expand its campus in the town as well as its work- force in the Bay State. Approximately 75 new employees will be hired to support clinical manufacturing at the fve-story, 175,000-square-foot facil- ity, which the company said is expected to begin operation in January 2019. n News Bioprocessing