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Elizabeth Rivera is a technical services manager at the Life Sciences Division of STERIS Corporation.
Dijana Hadziselimovic is a technical services laboratory specialist for the Life Sciences Division at STERIS.
Laboratory test methods evaluate cleaning agents and cleaning process design for removing resin residues from the surfaces of non-dedicated chromatography columns systems.
Liquid chromatography is used for separating materials in biopharmaceutical production, primarily for purifying proteins by separating product and impurities. The stationary phase in liquid chromatography uses fine, solid beads referred to as resins that are packed and held in a column by meshes. These particles can be physically or chemically modified to provide specificity to grab or repel molecules within mixtures.
Chromatography resins are typically dedicated to a single product. They can be either disposed of or cleaned to an acceptable level to render them suitable for use in subsequent cycles. The decision to reuse or dispose of resins is primarily driven by a cost analysis (1–2). For that reason, biopharmaceutical manufacturers reuse chromatography resins multiple times to make them affordable for inclusion in downstream processes (3–4). Regenerating or “cleaning” the resin is necessary for this purpose. The process consists of removing residual proteins and impurities from the resin while inside the column. Regeneration may be done after every loading cycle or after a few loading cycles.
Once impurities bind irreversibly, accumulating over time and consequently deteriorating the chromatography process performance, the resin needs to be regenerated to restore process performance and to minimize the risk of carryover (5). Caustic solutions at concentrations between 0.1–2 M were reported to be effective at regenerating most types of resins (6–7). Caustic solutions have also been effective at inactivating most viruses, bacteria, yeasts, fungi, and endotoxins and can be easily detected, removed, and disposed of. Other publications show that resins are effectively cleaned and sanitized with acidic solutions such as benzyl alcohol (8). Many times, the regenerating solution is used to store the cleaned resin for a prolonged time when not in use either in the column or in a separate storage vessel (7).
Regeneration of resin as described above has been well documented. Cleaning of the resin residue itself specifically from process equipment surfaces has not been widely addressed. While the resin packing is typically dedicated to one product, the chromatography column system may be employed for multiple products. After cleaning, the resins may be placed in another vessel for short or long-term storage. Other equipment that may have indirect contact with the resin are the slurry and packing tanks, and smaller parts such as hoses and valves. All these items must also be free from resin residues prior to use on the next product batch.
Most cleaning validation approaches are centered around removing either protein or process impurities from surfaces, and not on the resin residue itself. Residues from a chromatography resin are different from a protein in multiple ways. For example, the resin size may be more than 3000 times larger than a protein. As a general rule, the longer and more complex a molecule is, the harder it is to clean. Also, proteins in general degrade in the presence of caustic solutions while most resins have good chemical compatibility. The chemical compatibility allows resins to be stored in caustic solutions, which can be beneficial due to their antimicrobial properties. Lastly, carbon content is variable (but mostly negligible) from resin to resin compared to proteins.
For removing resin residues, the most commonly used solutions are sodium hydroxide (NaOH) and sodium chloride (NaCl), or even hot water for injection (WFI). Nonetheless, the physical and chemical properties of resins may be quite different from other residues of typical cell-culture processes. Cleanability studies should be conducted to demonstrate the suitability of these commodities for cleaning non-dedicated chromatography columns and to ensure that there is no cross-contamination between resins used for previously manufactured product into the next product. Cross-contamination concerns may also include microbial or allergen risks. This paper provides a case study that evaluates the cleaning efficacy of NaOH and formulated cleaning agents against resin residues. A general recommendation for cleaning resin residues is provided.
Defining the design inputs and outputs for cleaning resins is an important part of the cleaning process design. Cleaning parameters for a wash step may include the cleaning agent, concentration, temperature, time, cleaning method, water quality, and environmental factors (9). Cleaning agents should be selected based on laboratory studies that simulate the soil condition and cleaning method used as well as performing a supplier qualification and technical support review. A good experimental design must be used to identify the parameters that have a significant impact on cleaning within a selected range (10–11).
Manufacturing process parameters, such as dirty hold time, materials of construction, and soil conditions should be well understood before designing the cleaning process. Understanding all these factors will lead to a better design of the laboratory test model. As seen in Figure 1, laboratory testing can include coating of the soil onto a stainless coupon and conditioning it in an oven for a specified time and temperature (12). After the coupon is conditioned, it can be cleaned by several different cleaning methods.
In a laboratory set up, agitated immersion may be conducted as a standard for cleanability studies. Agitated immersion consists of the cleaning agent solution mixed in a beaker and equilibrated to temperature and concentration. The coupon is conditioned with the resin soil and placed into a beaker containing the cleaning agent. At select intervals, the coupon is visually inspected and either returned to the cleaning agent for additional time or evaluated for cleanliness using analytical methods, as needed. This cleaning method is generally considered worst-case when compared with clean-in-place systems because minimal action is employed.
The following discussion centers on the cleanability of various resins used and submitted by a biopharmaceutical company located in the United States. The biopharmaceutical site had concerns about the suitability of its current cleaning procedure using a commodity chemical (NaOH) for removing resin residues from the chromatography column and other ancillary equipment.
Laboratory evaluation and conditions. A total of seven different resins were evaluated: Q Sepharose XL Resin (GE Healthcare Life Sciences), SP Sepharose FF Resin (GE Healthcare Life Sciences), Butyl Sepharose HP Resin (GE Healthcare Lifesciences), ProSep vA Ultra Resin (MilliporeSigma), MabSelect SuRe Resin (GE Healthcare LifeSciences), Ceramic Hydroxyapatite Resin (Bio-Rad Laboratories), and Poros XS Resin (Thermo Fisher Scientific). The detailed test procedure is described in Table I and samples of resin-coated coupons are shown in Figure 2.
The critical parameters investigated during the cleaning process design testing included varying wash times, cleaning chemistries, cleaning agent concentration, and temperature (see Table II). The dirty hold time (air-dried for 48 hours and baked at 121 °C for one hour), cleaning action (low agitation, spray wash, and cascading flow), water quality (de-ionized), and surface characteristics (304 stainless steel with a 2B finish) were unchanged for this study. A coupon was considered clean if it was visually clean, water break free, and if the difference between its pre-coating weight and post-cleaning weight was not detectable (0.0 mg of residue) (13). Refer to Table II for a sample summary of study details.
Results and discussion. Sodium hydroxide (NaOH) is commonly adopted as the cleaning agent for removing resin residues from the surface. This cleaning agent primarily uses the mechanism of solubility of the solute in NaOH at the temperature cleaned.
When a formulated cleaning agent is used, the cleaning mechanisms to remove the residue from the surface may include solubility in an aqueous solution, wetting, emulsification, dispersion, chelation, and hydrolysis (14). These additional cleaning mechanisms are important in removing water insoluble residues from the surface. A formulated cleaner containing potassium hydroxide was successful in cleaning resin residues using 1% v/v cleaning solution at ambient temperature up to 60 °C depending on drying hold times of the coupons. Coated coupons, air-dried at ambient temperature for 48 hours, were easy to clean using the formulated cleaner (data results not shown). When coated coupons were baked at 121 °C for one hour, higher temperatures (45 °C to 60 °C) were required to clean the residues. Sodium hydroxide was not successful in cleaning the resins using elevated temperatures and longer cleaning times.
Deionized (DI) water and a formulated acid cleaner containing phosphoric acid also were not able to clean the residues using different cleaning parameters (refer to Table II). Cleaning results were confirmed by spray wash and cascading flow (data not shown).
A cleaning validation and changeover approach should consider resin removal from process equipment. The type of resin, temperature, and cleaning agent selection had a significant impact on cleanability of the stainless-steel coupons used in the study to evaluate the performance of various cleaning agents in removing residues of common chromatography resin residues from a stainless-steel surface. A potassium hydroxide-based formulated cleaning agent at 1% v/v up to 60 °C for up to 60 minutes was effective in cleaning the residues. Even though NaOH is widely used in the biopharmaceutical industry for cleaning resins, it did not perform as well as the potassium hydroxide-based formulated cleaning agent within this study. For some resins, a phosphoric acid-based cleaner was also effective in cleaning the residue and may be added as a secondary step particularly for mineral-based resins.
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7. PK Ng et al., Bioprocess International, pp 52–56 (May 2007).
8. M. Rogers, et al., Journal of Chromatography 1216, 4589–4596 (2009).
9. G. Verghese, “Selection of Cleaning Agents and Parameters for cGMP Processes,” Proceedings of the INTERPHEX Conf. (Philadelphia, PA, 1998), pp. 89–99.
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11. D. Hadziselimovic et al., Journal of GXP Compliance 16 (3) 32–38 (2012).
12. P. Lopolito, “Critical Cleaning for Pharmaceutical Applications”, in Handbook for Critical Cleaning Applications, Processes and Controls, B. Kanesgsberg and E. Kanesgsberg, Eds. (CRC Press, Taylor & Francis Group, 2nd Ed., 2011).
13. D. Hadziselimovic et al., Journal of GXP Compliance 16 (3) 32–38 (2012).
14. G. Verghese et al., “Cleaning Agents and Cleaning Chemistry,” in Cleaning and Cleaning Validation Volume I, P. Pluta, Ed. (Davis Healthcare International and Parenteral Drug Association, 2009), pp. 103–121.
Vol. 43, No. 4
When referring to this article, please cite it as E. Rivera and D. Hadziselimovic "Cleaning Chromatography Resin Residues from Surfaces," Pharmaceutical Technology 43 (4) 2019.
Elizabeth Rivera is a technical services manager, firstname.lastname@example.org; and Dijana Hadziselimovic is a technical services laboratory specialist, both at the Life Sciences Division of STERIS Corporation.