All the experiments in this study were conducted using a bench-scale cleaning model comprising the following components:
- Reciprocating shaking water bath: A Precision Scientific model 25 shaking water bath (Precision Scientific, Winchester, VA)
was used to control temperature and agitation. Oscillation frequency and amplitude were adjusted to obtain the desired linear
velocity. The temperature in the water bath was maintained with an accuracy of ±1°C.
- Stainless steel coupons: 6 x 3-inch stainless steel (304L) nonelectropolished coupons (Q-Lab Corp., Cleveland, OH) were used
in this study. Although the equipment used in biopharmaceutical manufacturing is typically made from 316-grade stainless steel,
for the purpose of these cleanability experiments, it was assumed that the grade of steel (304 versus 316) would not have
a significant effect on the relative cleanability of drug products. Differences in stainless steel surface finishes were not
included in the scope of this evaluation either.
- Cleaning agents: The cleaning solution used in this study was either de-ionized water or a CIP-100 solution (Steris, Mentor,
OH). In addition, CIP-200 (Steris) and the alcohol solution Septihol (Steris) were used for precleaning the coupons.
Four protein drug products (labeled as products A, B, E, and H in this article) were evaluated in these studies. Products
A and B are antibodies and products E and H are protein products. These aqueous products contained different excipients and
stabilizers, and were formulated at various concentrations (≥100 mg/mL).
Stainless steel coupons were precleaned in a cycle that comprised a CIP-100 wash, CIP-200 wash, and water for injection (WFI)
rinses, and dried before use. This precleaning procedure helped to cleanse any residual coating from the coupon fabrication
process and also ensured that all new coupons were in the same initial condition before starting the experiment. Of each protein
product, 250 μL was spotted on the coupon (with a maximum of six spots per coupon). Sufficient resolution was offered by the
250 μL to differentiate among the cleaning times of all four products in reasonable experiment duration. Each protein soil
was spread into a circular spot that was 1.35 cm in diameter. Variability in spot size and shape and in the measured cleaning
time was minimized by using new coupons for each evaluation. The spotted coupons were air dried at room temperature for 24
h before cleaning. It should be noted that the selection of 250 μL over a circular spot 1.35 cm in diameter might not be completely
representative of the typical product monolayer observed on the manufacturing equipment. As a result, the absolute cleaning
times reported in this study are not reflective of a large-scale cleaning cycle.
Before starting the cleaning experiment, the water bath tank was filled with the appropriate cleaning solution. This volume
was large enough (>1,000 times the protein load) to ensure that the performance of the cleaning process was not limited by
the amount of cleaning solution. The temperature and agitation speed of the water bath were set based on the selected cleaning
conditions. Based on the amplitude and frequency of the reciprocal shaking motion of the coupons, the cleaning fluid velocity
relative to the product spot was computed. The product spots on the coupons were monitored visually and the total time taken
by the spot to appear visually clean was taken as the cleaning time for that spot. At least three spots were studied for each
of the four products to obtain an estimate of standard error in the reported cleaning time. Visual inspection is an important
element of cleaning validation and offers a simple, noninvasive technique for surface cleanability assessment.6,12–15 If needed, the technique can be easily coupled with more sensitive assays (gravimetric or total organic carbon).