Process considerations related to the microencapsulation of plasmid DNA via ultrasonic atomization

An effective means of facilitating DNA vaccine delivery to antigen presenting cells is through biodegradable microspheres. Microspheres offer distinct advantages over other delivery technologies by providing release of DNA vaccine in its bioactive form in a controlled fashion. In this study, biodegradable poly(D,L-lactide-coglycolide) (PLGA) microspheres containing polyethylenimine (PEI) condensed plasmid DNA (pDNA) were prepared using a 40 kHz ultrasonic atomization system. Process synthesis parameters, which are important to the scale-up of microspheres that are suitable for nasal delivery (i.e., less than 20 μm), were studied. These parameters include polymer concentration; feed flowrate; volumetric ratio of polymer and pDNA-PEI (plasmid DNA-polyethylenimine) complexes; and nitrogen to phosphorous (N/P) ratio. PDNA encapsulation efficiencies were predominantly in the range 82-96%, and the mean sizes of the particle were between 6 and 15 μm. The ultrasonic synthesis method was shown to have excellent reproducibility. PEI affected morphology of the microspheres, as it induced the formation of porous particles that accelerate the release rate of pDNA. The PLGA microspheres displayed an in vitro release of pDNA of 95-99% within 30 days and demonstrated zero order release kinetics without an initial spike of pDNA. Agarose electrophoresis confirmed conservation of the supercoiled form of pDNA throughout the synthesis and in vitro release stages. It was concluded that ultrasonic atomization is an efficient technique to overcome the key obstacles in scaling-up the manufacture of encapsulated vaccine for clinical trials and ultimately, commercial applications.

Development of a pilot-scale bacterial fermentation for plasmid-based biopharmaceutical production using a stoichiometric medium

The recognition of the potential efficacy of plasmid DNA (pDNA) molecules as vectors in the treatment and prevention of emerging diseases has birthed the confidence to combat global pandemics. This is due to the close-to-zero safety concern associated with pDNA vectors compared to viral vectors in cell transfection and targeting. Considerable attention has been paid to the potential of pDNA vectors but comparatively less thought has been given to the practical challenges in producing large quantities to meet current rising demands. A pilot-scale fermentation scheme was developed by employing a stoichiometrically-designed growth medium whose exceptional plasmid yield performance was attested in a shake flask environment for pUC19 and pEGFP-N1 transformed into E. coliDH5α and E. coliJM109, respectively. Batch fermentation of E. coliDH5α-pUC19 employing the stoichiometric medium displayed a maximum plasmid volumetric and specific yield of 62.6 mg/L and 17.1 mg/g (mg plasmid/g dry cell weight), respectively. Fed-batch fermentation of E. coliDH5α-pUC19 on a glycerol substrate demonstrated one of the highest ever reported pilot-scale plasmid specific yield of 48.98 mg/g and a volumetric yield of 0.53 g/L. The attainment of high plasmid specific yields constitutes a decrease in plasmid manufacturing cost and enhances the effectiveness of downstream processes by reducing the proportion of intracellular impurities. The effect of step-rise temperature induction was also considered to maximize ColE1-origin plasmid replication.

Weight and weight change following breast cancer : evidence from a prospective, population-based, breast cancer cohort study

Background: While weight gain following breast cancer is considered common, results supporting these findings are dated. This work describes changes in body weight following breast cancer over 72 months, compares weight with normative data and explores whether weight changes over time are associated with personal, diagnostic, treatment or behavioral characteristics. Methods: A population-based sample of 287 Australian women diagnosed with early-stage invasive breast cancer was assessed prospectively at six, 12, 18 and 72 months post-surgery. Weight was clinically measured and linear mixed models were used to explore associations between weight and participant characteristics (collected via self-administered questionnaire). Those with BMI changes of one or more units were considered to have experienced clinically significant changes in weight. Results: More than half (57%) of participants were overweight or obese at 6 months post-surgery, and by 72 months post-surgery 68% of women were overweight or obese. Among those who gained more weight than age-matched norms, clinically significant weight gain between 6 and 18 months and 6 and 72 months post-surgery was observed in 24% and 39% of participants, respectively (median [range] weight gain: 3.9kg [2.0-11.3kg] and 5.2kg [0.6-28.7], respectively). Clinically-significant weight losses were observed in up to 24% of the sample (median [range] weight loss between 6 and 72 months post-surgery: -6.4kg [-1.9--24.6kg]). More extensive lymph node removal, being treated on the non-dominant side, receiving radiation therapy and lower physical activity levels at 6 months was associated with higher body weights post-breast cancer (group differences >3kg; all p<0.05). Conclusions: While average weight gain among breast cancer survivors in the long-term is small, subgroups of women experience greater gains linked with adverse health and above that experienced by age-matched counterparts. Weight change post-breast cancer is a contemporary public health issue and the integration of healthy weight education and support into standard breast cancer care has potential to significantly improve the length and quality of cancer survivorship.