Analysis of the 300K retentates (see Figure 2, step 4) by GC showed a general trend of lower total fatty acid/GCMP ratios
in lots that did not generate precipitation downstream (see Figure 4). No specific level could predict if a lot would precipitate,
but 30-40 % (w/w), could be used as an alert level. This interpretation was enhanced when the specific concentration of palmitic
acid entering the deacetylation/saponification process was examined. Deacetylations with palmitic acid >0.7 g/l were likely
to precipitate (see Table III).
Table IV: Fatty acid composition (%) at different stages of processing in lots that precipitated fatty acid.
The 300K retentate was the starting material for the saponification reaction. There is a mixture of saturated (palmitic, myristic)
and unsaturated (palmitoleic, oleic) fatty acids in this retentate (see Table IV). The precipitate in both the 50K and 30K
retentates was composed primarily of sodium palmitate. This contrast is explained by the differences in solubility of the
fatty acids. As reported by McBain et al9, the sodium salts of unsaturated fatty acids were more soluble than the sodium salts
of saturated fatty acids. Another factor that affects the fatty acid composition of the precipitate is the length of the fatty
acid hydrocarbon chain: the longer the hydrocarbon chain, the less soluble the fatty acid was in water. Sodium oleate and
sodium laurate are soluble in water at temperatures under 45°C. Sodium myristate, palmitate, and stearate have much lower
solubility at the same temperature. This property may be the reason for the oleic acid to be the more abundant unsaturated
fatty acid in the precipitate, despite the fact that palmitoleic acid is one of the most abundant fatty acid in the 300K retentate.
Since an increase in fatty acid concentration in the 300K retentate was associated with the use of a damaged pump, it is reasonable
to conclude that the damaged pump affected the integrity of the cells by increasing shear forces due to metal-to-metal contact.
The issue described here highlights the importance of preventive maintenance. The pump had been in place for several years
and appeared to work properly, in that it maintained normal flow rates and pressures during operation. Maintenance technicians
performed preventive maintenance at routine intervals, focusing on electric current demand and replacing hydraulic oil in
the gear case. The pump head was not routinely examined. Given what was learned about the sensitivity of bacterial cells to
shear, the authors now visually inspect the pump more often and replace it at any sign of damage.
The authors wish to thank Liqiong Fang, PhD, research scientist, Kirk Ashland, PhD, senior research scientist, Frank Hua,
PhD, research scientist, and Catherine Quinn, research associate, all at Baxter Healthcare, Round Lake. IL, who provided critical
analyses in support of this study.
AMY ROBINSON, PHD is a senior manager, SHWU-MAAN LEE,* PHD is a technical director, BOB KRUSE, PHD is a research scientist, and PEIFENG HU, PHD is a principal scientist, all at Baxter Healthcare, 8000 Virginia Manor Road, Suite 140. Beltsville, MD 20705. firstname.lastname@example.org
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