Purified water for pharmaceutical purposes must be free of microbial contamination and pyrogens. Even with the additional sanitary and disinfecting treatments applied to the system (sequential ...operational stages), Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were isolated and identified from a thirteen-stage purification system. To evaluate the efficacy of the chemical agents used in the disinfecting process along with those used to adjust chemical characteristics of the system, over the identified bacteria, the kinetic parameter of killing time (D-value) necessary to inactivate 90% of the initial bioburden (decimal reduction time) was experimentally determined.
Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were called in house (wild) bacteria. Pseudomonas diminuta ATCC 11568, Pseudomonas alcaligenes INCQS , Pseudomonas aeruginosa ATCC 15442, Pseudomonas fluorescens ATCC 3178, Pseudomonas picketti ATCC 5031, Bacillus subtilis ATCC 937 and Escherichia coli ATCC 25922 were used as 'standard' bacteria to evaluate resistance at 25 degrees C against either 0.5% citric acid, 0.5% hydrochloric acid, 70% ethanol, 0.5% sodium bisulfite, 0.4% sodium hydroxide, 0.5% sodium hypochlorite, or a mixture of 2.2% hydrogen peroxide (H2O2) and 0.45% peracetic acid.
The efficacy of the sanitizers varied with concentration and contact time to reduce decimal logarithmic (log10) population (n cycles). To kill 90% of the initial population (or one log10 cycle), the necessary time (D-value) was for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite, D = 7.9 min; (vii) mixture of hydrogen peroxide (2.2%) plus peracetic acid (0.45%), D = 5.5 min.
The contact time of 180 min of the system with the mixture of H2O2+ peracetic acid, a total theoretical reduction of 6 log10 cycles was attained in the water purified storage tank and distribution loop. The contact time between the water purification system (WPS) and the sanitary agents should be reviewed to reach sufficient bioburden reduction (over 6 log10).
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
► ATPMS are an interesting alternative for GFPuv extraction and LPS removal. ► ATPMS technology proved to be effective in GFPuv recovery. ► GFPuv is recovered into the micelle-poor phase, due to ...excluded-volume interactions. ► Triton X-114 can be applied for removal of higher LPS concentrations. ► ATPMS can be exploited as the first step for purification in biotechnology processes.
The viability of large-scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, since endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. This work aimed to study the use of aqueous two-phase micellar systems (ATPMS) from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv), which works as a biological indicator. The GFPuv extraction and LPS removal were evaluated in ATPMS, partition assays were carried out using pure GFPuv and cell lysate from
Escherichia
coli. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle-poor phase (
K
GFPuv
>
1), and LPS removal into the micelle-rich phase (%
REM
LPS
>
98%). GFPuv was partitioned preferentially into the micelle-poor phase due to excluded-volume interactions in the micelle-rich phase. Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations.
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