Cleaning cycle conditions of extreme pH and temperature (60°C
80°C) are known to result in protein degradation [1]. Traditional
maximum allowable carryover (MAC) assessments based on
acceptable or permissible daily exposure (ADE or PDE) assume
intact protein.
Thus, demonstrating the degree of degradation of the product can
aid in setting more meaningful acceptance limits for carryover into
the subsequent product.
The Hyde Analytical Laboratory was contracted to assess the effectiveness of the client’s current full-scale batch cleaning
and/or sterilization cycles in protein degradation for multiple products. Additionally, feasible cleaning conditions that could be
implemented at full-scale were evaluated to determine a cycle capable of complete degradation of the products.
Degradation of the product at small scale was studied by exposing the process soil to simulated cleaning and/or steaming
conditions. The degree of degradation was then assessed by subjecting the treated process soil to sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE), which allowed for visualization and quantification of the protein degradation, as
applicable.
Study
Overview
The laboratory conducted small-scale studies to
investigate cleaning cycle induced protein
degradation of multiple products.
The results aided the identification of a full-scale
cycle capable of protein degradation, as well as
the determination of an appropriate strategy for
setting carryover limits for patient and product
safety.
Analyzing protein degradation
Challenges in cleaning cycles
Scope
and Deliverables
Degradation Evaluation:
Determine the effectiveness of the current batch cleaning cycles in degrading the protein of interest. The current cleaning
cycle parameters included 1M NaOH at ambient temperature and various concentrations and temperatures of CIP-100.
Cycle Parameter Comparison:
Evaluate multiple cycle parameters to identify the set of inputs that is capable of effective degradation of the protein. This
included additional concentrations, temperatures, and durations of CIP-100 exposure that were feasible for implementation at
full-scale.
Full-Scale Recommendation:
Provide a recommendation for cleaning cycle parameters that effectively degrade the proteins either via routine cleaning or a
changeover cycle. Additionally, provide recommendations for an alternative approach to calculating MAC limits based on
degraded protein
Solutions, Results,
and Accomplishments
The current full-scale batch cycles do not achieve complete degradation of the products. The current cleaning cycle achieves partial degradation of the proteins, but not full degradation of the intact protein. Additionally, all degradants were above the UFDF membrane cut-off size and would be retained with the product during filtration.
Steaming does not aid the degradation of the proteins. Simulation of the current sterilization (steam) cycle did not result in effective degradation.
To achieve full degradation, the current batch cycle must be run in triplicate, or a changeover cycle is required. The changeover cycle consisted of a higher concentration and temperature of CIP-100 as compared to the current batch cycle, as well as a longer duration
A cleanability ranking matrix was generated. The matrix identified the soil found to be the hardest to clean, which can be leveraged to reduce full-scale validation activities.
