PLANIFICACIÓN DE LOS PLANES DE INVERSIÓN DE

Production of gene delivery complexes will require collection following mixing of the components and storage of large quantities of complexes. The possibilities for collection would include collection directly into small vials or, alternatively, bulk collection followed by transferral to small vials. There are advantages and disadvantages to each strategy in terms of contamination, requirement of additional mixing, process time and batch-to-batch variation. An assessment of the physical characteristics of DNA complexes collected under different conditions was made in order to assess the extent of influence exerted by these variables.

The collection method and the quantity collected were varied using collection vessels ranging from cuvettes, falcon tubes to glass beakers (Figure 3.7). A series of four experiments were performed in which complexes were mixed at a flow rate of lOOmlmin'^ and either the collection vessel or the amount of complexes collected in that vessel was varied. Within each of those four experiments between six and twelve samples were analysed to generate information regarding reproducibility. The complexes prepared in these experiments were formed from ctDNA and PLL at a charge ratio of +2.0 and a DNA concentration o f 25pgml‘^ in 20mM HEPES pH7.2. The average size data gained from the experiment (figure 5.1) demonstrates that bulk collection of the polyplex solution followed by the subsequent distribution of the complexes into smaller vials results in little or no variation as a function of the type of vessel used for collection of bulk solution or the quantity collected.

Chapter 5. Manufacture o f Non-viral DNA Complexes: Results and Discussion 200 ? 150 100 0 2 4 6 8 10 12 14 Sam p le number •C uvette sam p les

24m l c o m p le x e s - 2 0 0 m l B eaker S am p les

F alcon sam p lesiamp

50m l c o m p le x e s - 2 00m l B eaker S am p les

Figure 5.1. A verage particle size o f ctDNA/PLL 29300 complexes as a function o f collection m ethod and volume. The complexes were p rep a red at a charge ratio o f +2.0 in 20mM HEPES pH7.2, mixed at a flo w rate o f lOOmlmin^ and collected into cuvettes, falcon tubes o r a 200ml glass beaker.

H o w e v e r w hen co lle c tin g straig h t in to sm all vials, re p resen te d by cu v e tte s, the size o f the re su ltin g c o m p lex e s are slig h tly sm aller, at ap p ro x im a te ly 80nm , than those c o lle c te d in to falcon o r b ea k er v esse ls w hich had a size o f ap p ro x im a te ly lOOnm. F ig u re 5.1 also illu strates the high level o f re p ro d u c ib ility g a in e d usin g th e tw in sy rin g e p u m p m ix in g d evice, as there is little b atch to batch v ariatio n b etw e en th e sizes o f the c o m p lex e s in the tw elve re p lic ate sam p les p re p are d u n d e r fix ed collectio n co n d itio n s.

T h e c le a r d iffe ren c e betw een the sizes o f th o se co m p lex e s c o lle c te d on a larg e-scale, th at is, in to a falcon tube or glass beak er, and those co lle c te d on a sm all-sca le , into c u v e tte s, p ro d u c ed d o u b t o v er the q u ality o f m ix in g o cc u rrin g in th e m ix in g tube o f the sy rin g e pum p. T o assess these d iffe ren c es a tim e d titratio n e x p e rim e n t w as p erfo rm ed at sp ecific flow rates betw een Im lm in * an d bOmlmin ’. A 1 m o la r so lu tio n o f HCl acid, c o n ta in in g the in d icato r B ro m o cry so l b lu e (S ig m a A ld rich ), an d a 1 m olar so lu tio n o f the alkali S odium H y d ro x id e w ere placed in sep a rate sy rin g es and m ixed usin g the tw in syringe pum p. T he tim e taken fo r the tw o so lu tio n s to m ix in the m ix in g

Chapter 5._______________________M anufacture o f Non-viral DNA Complexes: Results and Discussion

tube to equilibrium, signalled by a change in the colour of the indicator, was recorded visually and used to assess the quality of mixing.

The results of the experiment indicated that acid and alkali neutralisation, specifically equilibrium, was reached prior to the solution exiting the mixing tube and therefore mixing quality in the device was of a high standard. Results of this test are not shown here as the colour change was virtually immediate, occurring within milliseconds of the two solutions mixing at the T-junction making it difficult to record precise times. According to the results gained in this investigation the method o f collection does affect the physical characteristics o f the resulting complexes while the amount collected appeared to have no affect. Complexes collected into small vials were, on average, 20nm smaller than those initially collected into larger vessel and subsequently divided. As the quality of mixing using the twin syringe pump has been shown to be of a very high standard it is likely that the reason for the difference in size is due to differences in process time, that is the time between collection and measurement, for example, the collection of 12ml followed by separation into six 2ml vials is significantly longer than the time taken to collect 2ml straight into a small vial. It therefore appears that even a short amount of time can be crucial and will affect the size o f the complexes. However, using this delivery systems the difference is not great enough to rule out either of the collection option and if this trend proved to be applicable to other systems it is likely that the choice is more likely to come down to other factors such as speed, contamination, control and reproducibility. Subsequent to this experiment all samples collected for size measurement were collected straight into cuvettes for immediate transfer into the zetasizer.