Static exposure to 0.5M NaOH showed mixed results. Gram-negative bacteria are easy to kill. Bacillus subtilis spores are unaffected by NaOH. Staphylococcus aureus, Candida albicans, and A. niger are initially inhibited by 0.5M NaOH, but stabilize. Heat treatment at 50°C compared with ambient trials showed increased
inhibition of C. albicans than with 0.5M NaOH. At similar inoculum concentrations, all of the controls exhibited < ± 3.0% change when heat was combined
with NaOH treatment. Destruction of B. subtilis spores was increased, but S. aureus showed enhanced resistance at 50°C, possibly a stress response.
Reducing S. aureus concentrations from 107.5 to 105.5 leads to increased inactivation kinetics. Protective effects of S. aureus glycocalyx and culture-derived flocculation were examined and were not a factor.
Kinetic reaction studies were conducted. Gram-negative bacteria were killed rapidly with 0.5 M NaOH. Spores cannot be effectively
cleaned with sodium hydroxide. Elevated temperatures (50°C) do not offer a clear cleaning advantage for the trialed organisms.
Static application of 0.5 M NaOH may not provide adequate removal of non-gram negative species and is very time dependent.
The process was tested at scale up using a Vantage L (32 x 250 mm) column and Matrex celluline CG700 chromatography media
with a packed bed height of 5.0 cm. Cleaning procedures included forward and reverse flow for 4 hours with both 0.5M and 1.0M
NaOH used. Four samples were taken during the water rinse phase. Long static soak proved insufficient; dynamic flow with direction
reversal is beneficial to the effectiveness of the sanitization process. 1.0 M NaOH was required to sanitize the Vantage L
column and spores.
Figure 4. Challenge bacteria concentration after various testing stages.
The final experimentally derived cleaning SOP was as follows:
- One column volume (CV) (plus pipe hold-up volume) of water is circulated at 100 cm/hr in reverse flow;
- Two CV of 1.0M NaOH is circulated at 100 cm/hr in reverse flow;
- One CV of 1.0M NaOH is pumped through the column at the same linear velocity in forward flow;
- Two CV of 1.0M NaOH (total) is recirculated for two hours at 100 cm/hr.;
- Neutralization: Water is flushed through the column at 100 cm/hr until the effluent pH is < 8.0.
- Total microbial exposure time to a pH of 12–14 in less than 3 hr.
Microbial challenge scale-up. To demonstrate that the column design is readily cleanable, the column was challenged with an extremely concentrated microbial
cocktail, subsequently cleaned, and validated, as follows.
- Column: Acrylic 450 mm glass 100 mm QuikScale column
- Medium: Matrex Cellufine CG700 resin
- Cleaning agent: 1.0 M NaOH
- Microbes: E. coli, S. aureus, P. aeruginosa, C. albicans
- Initial inoculum concentration: 2.68 x 108 culture (static phase), (-1 x 1012 total cells loaded)
- Breakthrough concentration: 960 x 107 clm/mt — stationary
• Inoculate column with high concentrations of micro-organisms.
• Incubate column for 18 hr.
• Perform sanitization procedure.
• Following neutralization, analyze column with ortho-gonal methods.
- Enumeration: spread plating
- Acceptance criteria: (USP26-NF21S1 monograph <1231>). Water for Pharmaceutical Purposes states not more than 10 CFU per 100
mL WFI may be present. Very low levels of post cleaning microbial presence are necessary to prove that the column can be readily
- Swab locations (according to Le Blanc):3
• Calcium alginate in trypticase soy broth (TSB), rolling biodirectional > 1 cm2
- Turbidity of rinse
- Membrane filtration of rinse and plating of membrane
- Desorbtion of cells from quantified amount of chromatography media
- Immersion of column seals and gaskets in liquid broth
Results. No growth of the spiked organism present in the original cocktail was observed in the rinse samples (LOD = 1 CFU/100 mL).
All swabs and chromatography media samples (pour plating and liquid desorption) were negative for growth. Application of the
developed cleaning protocol significantly reduced populations of microorganisms. Rigorous and redundant examination procedures
provided several metrics that can be used to support the claim of sanitary design (Figures 4 and 5).
Figure 5. The cleaning efficacy of the column was determined through surface swabs on different column wetted parts, then
analyzed for microbial growth.
CONCLUSIONS OF COLUMN CLEANABILITY CASE STUDY
- 1.0 M NaOH is a potent anti-microbial agent.
- Efficacy is greatly enhanced by dynamic rinsing and recirculation.
- Contact time >2 hr is recommended for maximum sanitization.
- Sanitization procedures can be effectively conducted at room temperatures.
- Gram negative bacteria are killed rapidly.
- All cleaning should be preceded by a 1 CV flush with sterile buffer.
- Pour-plating is inferior to a discrete desorption step because of the high turbidity of solidified agar on chromatography
media plate, which makes it difficult to resolve colonies.
- Background flora necessitate controlled environment for challenge studies.
- These results showed that the column met the metrics of cleanability described.
The columns proved to be cleanable with a variety of challenge materials in extremely contaminated conditions. The investigation
allowed design changes to be made that not only improved the system, but also provided quantifiable evidence that the final
product could be cleaned sufficiently in typical user scenarios.
Because each cleaning process is different and presents unique challenges, cleaning protocols and agents must be selected
on the nature of the contaminants, media, equipment, and the level of sanitization required. Ultimately, the pharmaceutical
company is responsible for validating its own process. However, purchasing equipment that has been tested with rigorous scaled-down
cleaning trials helps the company develop and test its own processes with confidence.
The author thanks Aaron Noyes, formerly of Millipore, for his contribution to this research.
Chris Antoniou is group R&D manager and Hilary Carter is development engineer, both in the BioPharmaceutical Division, Millipore Corporation, 80 Ashby Road, Bedford, MA 01730, Tel: 781.533.2650,
Fax: 781.533.3134, email@example.com
1. Lindsay, J. Cleaning and Cleaning Validation: A Biotechnology Perspective, PDA, 1996.
2. U.S. Pharmacopeia, 26 / National Formulatory 21, S1
3. LeBlanc, DA. Sampling, Analyzing, and Removing Surface Residues Found in Pharmaceutical Manufacturing Equipment. Microcontamination 1993 May: 37-40.