(W1130-11-63) A New Shear Protectant: Introducing Kolliphor^® P188 Cell Culture

Purpose: In the past decades, the bioprocessing industry has made significant technological strides that have led to higher cell densities and more final drug product output¹. Raw materials in cell media play a fundamental role in running bioprocesses that are both efficient and high yielding. Reagents in media, and buffers make up a great part of the Cost of Goods of a bioprocess and underperforming raw materials can lead to costly disruptions^2,3. As the complexity and intensity of bioprocesses continues to grow, efforts in identifying which raw materials are introducing irregularities and their impact are being evaluated across the industry. Bioprocess interruptions or failures have an impact not only on economic but also sustainability goals^4,5. While the overall pharmaceutical industry has made strides on minimizing environmental impact, assessments in the biologics industry are in their infancy⁴. A strategy to meet sustainability goals is to run efficient and reproducible bioprocesses where reliable raw materials play a crucial role in minimizing bioprocess anomalies^4. A critical raw material, poloxamer 188, has a history of impacting cell performance due to hydrophobic⁶ and low molecular weight species⁷. Differences in average molecular weight have been shown to have an impact in cell performance⁸ and foaming⁹. The present work looks to showcase the performance of the new, lower molecular weight Kolliphor^® P188 Cell Culture (Fig. 1). This new shear protectant is a GMP material for bioprocessing applications where a lower average molecular weight poloxamer could be beneficial.

Methods: Three commercial lots of Kolliphor^® P188 Bio (P188 Bio) and Kolliphor^® P188 Cell Culture (P188 CC) were tested for shear protection performance using a cell culture test. Briefly, 250mL baffled flasks were seeded at a density of 3 x 10⁵ cells/mL using CHO-S cells cultured in chemically defined medium with 0.25g/mL of poloxamer 188. Flasks were incubated at 37°C, 5% CO₂ at a 300 RPM shaking intensity over 3 days. Viable cell density was measured daily and used to calculate cell viability. Samples were tested in triplicate.Three commercial lots of P188 Bio and P188 CC were tested for foam stability. Briefly, 50 ml of 0.3% solutions of poloxamer 188 were heated to 37°C. Using a sparger, foam was created until it reached a height of >100mm. The foam height was measured until the foam height collapsed to < 60mm. Samples were tested in triplicate. To calculate foam rate of decay, the following equation was used: Rate of Decay = (maximum height of foam - final height of foam)/time of decay.

Results: Cell performance was analyzed using analysis of variance (ANOVA), where we compared cell performance, using cell viability and viable cell number as parameters, of commercial lots of Kolliphor^® P188 Bio and Kolliphor^® P188 Cell Culture to the positive control (Fig. 2). When comparing lots in high shear conditions, we found Kolliphor^® P188 Bio lots had good performance indicated by high cell viability and viable cell count when compared to the positive control. Lots of Kolliphor^® P188 Cell Culture showed similar cell viability values to the positive control and samples of Kolliphor^® P188 Bio but a higher, statistically significant viable cell density (Fig. 2). Foam stability was examined using a dynamic foam analyzer (Fig. 3) where samples of LMW and HMW poloxamer 188 were compared. The foam stability of the LMW samples is less, making the foam height dissipate faster than the HMW sample (Fig. 3). We did not find these differences to be statistically significant.

Conclusion: In conclusion, Kolliphor® P188 Bio demonstrates excellent shear protection, resulting in high cell viability and viable cell density. Additionally, Kolliphor® P188 Cell Culture has shown to positively impact viable cell density under high shear conditions. Furthermore, the lower molecular weight of Poloxamer 188 in Kolliphor® P188 Cell Culture leads to less stable, faster-dissipating foam, which can be beneficial in specific bioprocessing applications.

References: [1] Dryden, W.A et. al. 2021, Biochem Eng Jour 167:107892
[2] Dickens, J. et al. 2018 Curr Op Chem Eng 22: 236-243
[3] Mahal, H. et. al. 2021. Biotech. Bioeng. 118:3468-3485
[4] Madabhushi, S. et. al. 2018. Biotechnol. Prog. 34 (6): 1566-73
[5] Budzinski, K., et al. N Biotechnol. 49, 2019: 37-42
[6] Chang, D. et. al. 2017. Colloids Surfaces156:358-65
[7] Bandyopadhhyay, A. et. al. 2022. J Biotech 351:13-22
[8] Safta, J. et. al. 2024 Biochem Eng J 202: 109146
[9] Zhang, S. et. al. 1992. J Biotech 25:289-306

Figure 1. Average molecular weight targets for Kolliphor® P188 Bio and Kolliphor® P188 Cell Culture

Figure 2. 3-day cell viability (circles) and viable cell density (columns) between Kolliphor®P188 P Bio and Kolliphor®P188 Cell Culture

Figure 3. Foam decay of 3 commercial lots of Kolliphor® P188 Bio (green) and Kolliphor® P188 Cell Culture (blue) with CHO-S cells at 0.1% inclusion