Design of Experiments: Single Parameter Evaluation
Table 1. List of process operating parameters and the operating ranges for large-scale and bench-scale cleaning process
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The key parameters explored in this evaluation included: temperature, concentration of cleaning solution, dirty hold time,
and agitation during cleaning. The first phase of this study was designed to identify the parameters that have a significant
impact on the cleaning process while minimizing cross interactions among input variables. Each parameter was varied within
a specified range that was preselected to be much larger than the normal operating range. One parameter was varied while others
were kept constant at the baseline cleaning conditions. These baseline conditions were selected to mimic the manufacturing
cleaning cycle. Table 1 shows the baseline cleaning conditions and the selected evaluation range for each parameter.
Augmented Design of Experiments: Cross-Interaction Among Parameters
After the first phase of experiment was completed, an augmented design of experiments was constructed using the JMP statistical
software (SAS, Cary, NC), and additional experiments were conducted to assess the effect of variable cross interactions on
the cleaning process.16 A leverage plot analysis showed that the cross-interactions were limited to two parameters: concentration and temperature
of the cleaning solution.
RESULTS AND DISCUSSION
Key Operating Parameters
Figure 1. Relationship between cleaning time and various key operating parameters: (a) temperature, (b) CIP-100 concentration,
(c) dirty hold time and (d) agitation. Only one parameter was changed at a time, while others were kept constant at the baseline
conditions listed in Table 1.
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Temperature. The conventional approach to cleaning processes may drive us to believe that it is always better to use a cleaning solution
at a higher temperature. Although this may be the case for products where solubility increases with temperature (mainly small-molecule
based pharmaceuticals), protein products exhibit a different trend. Figure 1a shows how the cleaning time changed for four
products as the temperature of the 1% v/v CIP-100 solution was increased while keeping other operating parameters the same
as the baseline listed in Table 1. The longest cleaning time occurred at 55°C. Interestingly, the shortest cleaning time for
all products is observed at the lower temperatures. We attribute this trend to the unique behavior of proteins, for which
cleaning time is a combination of two competing phenomena: the dissolution of protein soil in the cleaning solution (controlled
by solubility, wetability, etc.), and protein degradation under high pH and temperature conditions though a series of chemical
reactions including hydrolysis, oxidation, and de-amidation. Protein solubility is maximized in the lower temperature range
(20–30°C) where protein molecules are dissolved in the aqueous solution while maintaining their structure. As the temperature
increases, protein products lose their structure, start to denature, stick to the surface, and become increasingly more difficult
to dissolve. The onset of denaturation is driven by the melting temperature of each product. Product H, with the lowest denaturation
temperature, shows an increase in cleaning time even for a temperature of 40°C. As the temperature is increased, CIP-100 solution
(pH >11) also starts to degrade the product molecules into smaller fragments.6,17 At an intermediate temperature of approximately 55°C, although protein degradation has started, it is the protein denaturation
phenomenon that dominates, resulting in an overall increase in the cleaning time, the effect being more noticeable for antibody
products (A and B).
As the temperature increases beyond 70 °C, the alkaline solution degrades the protein molecules more effectively into smaller
fragments and also enhances protein removal from the surface. It is therefore concluded that although protein dissolution
is high near ambient temperatures, a temperature of ≥70°C is needed to degrade the protein molecules into smaller fragments.
Cleaning validation may seek product degradation as a requirement in addition to product removal from surfaces because smaller
peptides can be considered less immunogenic than native product.18
Similar experiments conducted with cleaning in de-ionized water at various temperatures also showed ambient-temperature water
resulted in shorter cleaning times for all four products. Higher temperatures (≥55°C) caused protein denaturation, making
the cleaning solution (water) turbid. In the absence of degradation action of the CIP-100 solution, none of the four products
could be cleaned within the experimental duration of 2 h when subjected to water at 70°C. It is therefore recommended that
the prerinse step, often used before the equipment hold, should be performed using ambient-temperature water to remove the
majority of the protein load from the equipment.
CIP-100 concentration. The concentration of the cleaning agent also plays a critical role in governing the ability of the cleaning fluid to remove
protein products from equipment surface. Although high temperatures favor denaturation (loss of secondary and tertiary structure),
a higher CIP-100 concentration increases the OH¯ concentration, which results in increased degradation rates. The primary
effect of alkaline agents is peptide bond hydrolysis, which results in protein molecules being broken down into smaller peptide
fragments.
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