Bench-Scale Characterization of Cleaning Process Design Space for Biopharmaceuticals - A method to evaluate the relative cleanability of new products. - BioPharm International


Bench-Scale Characterization of Cleaning Process Design Space for Biopharmaceuticals
A method to evaluate the relative cleanability of new products.

BioPharm International
Volume 22, Issue 3

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
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.


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.
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 55C. 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 (20–30C) 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 40C. 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 55C, 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 ≥70C 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 (≥55C) 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 70C. 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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