The accuracy (percent difference between both the expected and measured TOC value) was calculated as follows:
% difference = (measured concentration – expected standard concentration/expected standard concentration) x 100.
Method linearity was verified using the sucrose standard at three concentrations, 250, 500, and 750 ppb of TOC (for each concentration
with three replicates). The correlation coefficient (R2) was calculated as follows:
in which X is the certified values of TOC standards and Y is the measured values of TOC standards (blank–corrected).
The limits of detection and quantitation (LOD and LOQ, respectively) were calculated using three sucrose standards at 250,
500, and 750 ppb of TOC (for each concentration with three replicates). Using a least squares linear regression algorithm,
the equation of the line relating the SDs of the standards to the actual measured concentrations of the standards was determined.
The y-intercept represents the SD at zero ppb of standard sucrose concentration. The LOD was the yintercept value multiplied
by 3, and the LOQ was the y-intercept value multiplied by 10.
Sample Preparation for Determining the Correlation Between TOC and Microorganisms
Both bacterial and yeast broths were grown until an optical density of 0.6 was reached, at 500 nm and 600 nm, respectively.
The culture media used to grow bacteria was LB (5 g/L yeast extract, 10 g/L triptone, 10 g/L NaCl). The culture media used
for yeast was YPD (20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract). These cell suspensions were centrifuged in a
TJ-6 centrifuge (Beckman Instrument, Fullerton, CA) for 10 min at 1,000g. The pellets were washed three times and suspended with low TOC purified water. The serial dilutions for wet cell concentrations
shown in Figure 1 also were prepared using the same purified water.
The coupons used in this work were representative of the materials of construction of the manufacturing equipment.
Therefore, both 100 cm2 stainless steel (316 L) and 100 cm2 borosilicate coupons were used in the swab recovery study. One side of each coupon was spiked with a solution (or biologic
sample) of cells or pure recombinant proteins, respectively. For bacterial and yeast cells, 200 μg, were applied. In the case
of recombinant proteins, 1.2 mg of SK, 100 μg of EGF, 100 μg of IFNα, and 12.5 mg of IFNα–PEG, were applied. The coupons were
allowed to dry completely for 4 h at 25 °C. A swab was moistened with low TOC water and the spiked coupon surfaces were swabbed
both vertically and horizontally. The swab end was cut off and placed in a vial with 40-mL of TOC water. The vials were capped,
shaked vigorously by inversion, and the TOC analysis was performed. The same quantity of each analyzed biologic sample was
spiked directly in 40-mL of TOC water (reference control).
Manufacturing Equipment Cleaning
The cleaning process was performed using sodium hydroxide and orthophosphoric acid, as described by Verghese10 and Mollah,5 following the standard procedures previously developed by the manufacturing department and approved by quality assurance
staff of the Center for Genetic Engineering and Biotechnology (CIGB).11–13 The final rinses of the fermentation and chromatography equipment were performed with purified water and water for injection