Kolliphor P 188 Bio Product Provides Protection Against Shear Stress in Biopharmaceutical Production

Poloxamer 188 is a pharmaceutical excipient used in topical, oral and parenteral applications. Beyond this, poloxamer 188 is known as a highly effective protective agent against shear stress and was used in the production of cell cultures in suspension form in the late 1980s. Due to the extreme sensitivity of CHO cell lines to shear stress forces, poloxamer 188 raw material is frequently preferred as a protective agent in the commercial production of monoclonal antibodies and therapeutic proteins. Even the use of poloxamer 188 at only 0.1% concentration shows positive effects on cell viability and growth rate. In recent years, some users in the biopharmaceutical industry have reported problems related to variable performance of the poloxamer 188 product, which has also created issues in meeting the requirements of relevant monographs in pharmacopeias. Raw materials with variable performance reduce protection of cells from shear stress in commercial production and lower cellular product titers. BASF initially marketed poloxamer 188 raw material as an excipient for oral or topical drugs. Following research into the use of poloxamer 188 in biological systems and collaborations with certain industry users, a systematic investigation was launched to identify the root cause of variable performance encountered in cell culture production. A specific type of poloxamer 188 with improved and consistent biological performance for cell culture production was developed through the application of necessary controls in light of the experience and knowledge gained. The newly developed poloxamer 188 raw material was specifically designed for cell culture applications. A validated cleaning procedure and production method were designed to reduce the risk of variable performance product, taking into account the needs of users in the biotechnology industry, and analytical developments were made in cell culture tests and new chromatographic methods. The experimental data and information mentioned were organized based on the technical document published by Felicitas Guth, Bastiaan Staal and Tonya Jackson.[1] Poloxamer 188 is a terpolymer with an average molecular weight of 8,400 Da composed of three blocks. As shown in Figure 1, ethylene oxide blocks are located on the right and left of the polypropylene oxide structure in the middle section. The raw material is produced by anionic polymerization using propylene oxide and ethylene oxide as monomers, diethylene glycol as initiator and alkali hydroxide as catalyst. Quality standards as a pharmaceutical excipient are defined in the European Pharmacopoeia[2], United States Pharmacopeia[3] and Japanese Pharmaceutical Excipients document[4]. Physicochemical properties are shared in Table 1.[2,3,5] Poloxamer 188 raw material produced by BASF is marketed under the trade name Kolliphor P 188. Poloxamer 188 is a non-ionic surfactant that does not cause irritation and forms relatively large aggregates rather than typical small micelles in aqueous solutions.[6] Many publications discuss the tendency of mammalian cell membranes to interact with polymers, and recommend solutions that provide stabilization under stress conditions.[7] Publications on systematic evaluation of the protective effect of poloxamer 188 in cell culture production first appeared in the 1990s. Shear stress occurring in the bioreactor during cell culture production results from mixing and oxygen feeding. Mechanical forces created by oxygen bubbles rising and bursting at the liquid-gas interface during aeration cause cell lysis.[8,9,10] Physical, mechanical and biological mechanisms have been proposed to explain the stabilizer and beneficial effect of poloxamer 188 on mammalian cells. According to some researchers, when poloxamer 188 is used at a concentration of 1 g/L in the medium, the tension at the liquid-gas interface decreases and the interaction between gas bubbles and cells is reduced. Consequently, cells adhere less to rising bubbles in the reactor and are trapped less in foam accumulating at the top, thus being better protected from mechanical forces.[11] The surfactant effect of poloxamer 188 has been shown to reduce the size of oxygen bubbles produced and thus have a positive effect on oxygen mass transfer.[12] The polymer has been observed to have high affinity for cell membranes and is thought to contribute to physical stabilization of the cell by forming a hydrophilic layer. Since this effect does not persist after poloxamer is removed, it is unlikely to have a long-term biological effect.[13] Although there is no general usage recommendation regarding how much to increase poloxamer 188 concentration, polymer use between 1-3%[12,14] and even at 5%[15] has been reported to provide advantages for certain reactor types and test conditions. The optimal amount can be determined specifically for the process and in this process, not only upstream process requirements but also downstream purification processes should be considered. Other poloxamer types[16], polyethylene glycol[10], methyl cellulose[17], or alkyl maltopyranoside[18,19], have been tested and recommended in this context. However, some have shown worse performance compared to poloxamer 188 and others have not provided commercial added value. Poloxamer 188 has become one of the important components of culture media for mammalian cells. In particular, it has become an even more important component for sensitive Chinese hamster ovary (CHO) cell lines frequently used in the production of therapeutic proteins and monoclonal antibodies. Recent advances in process and cell line development have enabled achievement of high cell densities and increased product titers to 10-13 g/L levels in commercial production.[20] Under these conditions, poloxamer 188 has had a significant effect in reducing shear stress forces and preventing cell aggregation. Thus, high rates of cell viability and growth rates have been achieved. Recently, it has been reported that variability in the performance of poloxamer 188 types used in oral drug applications has had a reverse effect on cell viability and product yield in commercial production of biological products. For this reason, the use of poloxamer 188 specifically developed for cell culture with consistent performance has become important and many test methods have been proposed that could predict this performance. Tharmalingam, T.[15] and Murhammar, D.W.[16], recommended carrying out small-scale cell culture experiments by generating shear stress through mixing operations for performance evaluation. In this context, the protective effect of poloxamer 188 was determined based on total viable cell count or growth rates following specified endpoints. These test systems were found to have the capacity to detect differences between different poloxamer 188 batches and the results obtained showed correlation with industry-scale applications. In addition to other performance methods such as cell culture and foam stability,[21] many chromatographic procedures have been published to provide information on polymer stability and composition and to show characteristic properties of poloxamer 188 raw material that could trigger performance variability in cell culture. In this context, reversed-phase liquid chromatography methods combined with systems of different sensitivities such as size exclusion chromatography combined with laser desorption ionization or nuclear magnetic resonance[22] and mass spectrometry[23] are used. A general consensus has emerged that very small amounts of hydrophobic and high molecular weight types that may be present in poloxamer 188 raw material could reduce protective effectiveness against shear stress.[21,24,25]

3. Detection of Impurities Using New Chromatographic Methods

BASF utilizes a new high-performance liquid chromatography method for the determination of hydrophobic types thought to have negative effects on cell culture performance. Gradient polymer elution chromatography (GPEC), operating in reversed-phase mode with a corona detector, enables detection of hydrophobic types present at levels below the limits found suitable for other poloxamer 188 types. Initially, gradient elution was started with a polar solvent, then switched to a non-polar solvent to increase polymer solubility, and separation was performed using reversed-phase (RP) mode with an apolar stationary phase. With the aforementioned method, very low-level hydrophobic types in the poloxamer 188 polymer can be determined. BASF has analyzed many different poloxamer 188 batches with the new RP-HPLC method. As shown in Figure 2, it was found that the presence of hydrophobic types varied between batches.

4. Use of the New Poloxamer Type in Biotechnology Applications

Based on experience gained during development work, BASF designed a new high-quality and high-purity poloxamer 188 product with the trade name Kolliphor P 188 Bio for biotechnology applications. It is considered that the application of additional controls such as microbiological tests, cell culture tests and RP-HPLC analysis and a validated production process in the production of Kolliphor P 188 Bio could enable compliance with certain quality requirements for biopharmaceutical production. Figure 3 shows RP-HPLC analysis results for three different batches of Kolliphor P 188 Bio and Figure 4 shows cell culture quantification results for three different batches of Kolliphor P 188 Bio. Following the analyses conducted, no hydrophobic peaks were detected in any of the three batches and the cell culture quantification results were found to be similar. Although used in low concentrations, poloxamer 188 raw material provides benefits in biopharmaceutical production by protecting mammalian cells from shear stress occurring in bioreactors. The latest publications show that poloxamer 188 exhibits variable performance due to the presence of very low levels of hydrophobic types not detectable by monograph methods. Based on this point, the Kolliphor P 188 Bio product was specifically designed for biotechnology applications with appropriate controls applied to ensure consistent performance.  

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