January 1, 2016 (Vol. 36, No. 1)
Nikolai Stankiewicz Ph.D. Head of Technology Transfer Laboratory Merck Millipore KGaA
Novel Technology Application Enhances Efficiency of Dry Power Formulations
Dry-powder cell-culture media (CCM) offer many advantages with respect to shipping and storage due to their reduced volume and increased stability. However, such media need to be highly soluble and easy to handle. This can be achieved by dry granulation, the agglomeration of fine CCM powders into larger granules using compression force only, that is, by means of roller compaction without water or other additives.
Although roller compaction is well established in pharma formulation, it is new in mammalian CCM. Regardless, roller-compacted granules produced from homogenous dry powders of different CCM formulations have been evaluated for handling efficiency and cell culture performance.
In this article, the efficiency enhancement will be evaluated by physicochemical methods such as bulk density and dust formation measurement as well as solubilization experiments. Abrasion behavior will be introduced as a quality measure for compacted CCM, and the practicality of compaction will be shown with growth, titer, and critical product-quality attribute data from a scale-down fed-batch cell-culture experiment.
This comprehensive dataset on compaction shows that this new technology application can help overcome drawbacks of dry powder CCM in bioprocessing.
For this study, different dry-powder CCM formulations were compacted using an RC-100 roller compactor (Powtec Maschinen und Engineering). Break energy was measured with a Revolution Powder Analyzer (PS Prozesstechnik), and dust formation was quantified using an IAQ-11-R Aerosol Spectrometer (GRIMM Aerosol Technik).
UPLC was used for amino acid quantification. Amino acids were first converted into stable derivatives using the AccQ-Tag™ reagent (Waters) then separated via UV-detector-coupled reverse-phase chromatography. Vitamins were quantified using an UHPLC-MS/MS assay.
For the solubility experiments, an SR0700ODLS bioreactor (DASGIP) with two Rushton impellers was used, and turbidity measured with a Turbiquant® 1500 IR turbidimeter (EMD Millipore). Complete dissolution is reached at a reading of <3 NTUs (nephelometric turbidity units).
Cell-culture fed-batch experiments were conducted in 30 mL of medium in 50 mL shake tubes, with feeding every second day. Cell density was measured using the Vi-CELL® instrument (Beckman Coulter), and titer was determined using the Biacore C instrument (GE Healthcare). Glycosylation was determined using CGE-LIF technology (Thermo Fisher Scientific), and charge variants were measured using the iCE280 system (ProteinSimple).
Compacted Media Integrity and Quality
Compacted CCM must have the same cell culture properties as the uncompacted powder. To verify this, the concentrations of selected CCM raw materials within the formulation were analyzed. The vitamin content measurements in Cellvento™ Feed-210 are compared to powder values in Figure 1. If the values determined for the powder are taken as 100%, the results indicate that compaction did not lead to depletion or degradation of vitamins.
Within a measurement accuracy of 15%, a maximum 12% decrease in vitamin concentration cannot be considered significant (Figure 1A). Similar results were obtained for amino acid quantification (data not shown). The homogeneity of compacted Cellvento Feed-210 was evaluated by measuring amino acid, metal ion, and vitamin content. All measurements performed on random samples from a 0.5 kg batch showed less than a 10% deviation from the mean, proving the homogeneity of the compacted feed formulation (data not shown).
Quality was evaluated by abrasion measurement, where abrasion was increased by adding ceramic balls to the abrasion-drum to address differences in quality and stability. This work demonstrated that the quality of compacted media depends on the formulation (Figure 1B).
The data in Figure 1A demonstrate that compaction does not alter the content of critical CCM components. Nevertheless, compaction is a formulation-dependent technology that has to be adjusted according to the CCM composition. For instance, elevated compaction pressure leads to a more stable product in media but not in feed formulations, correlating with their different amino acid levels.
Compared to dry-powder CCM, compacted media offer advantages that can improve shipping efficiency and risk mitigation in the media preparation process. For example, compaction generally reduces bulk volume, as indicated by an analysis of the bulk density of multiple formulations (Figure 2A). However, a formulation dependency ranging from a 1.2- to a 2.1-fold bulk density reduction can be observed accordingly.
Flowability was measured by determining the energy required to bring the CCM into free flow. Compaction reduces this energy by 1.7 to 8.6 kJ/kg, leading to easy flowing behavior compared to the powder (data not shown).
A major disadvantage of using fine dry media is dust—a hazard to media preparation workers that requires protective measures. The dust is also a potential contamination source. It may come into contact with production plant’s cleaning water, and it may not be fully poured away. For Cellvento CHO-210 compacted medium, up to a 12-fold reduction in dust formation was achieved (Figure 2B).
The dry CCM formulation data reveal that using compacted CCM can make dry-powder media applications more efficient to ship and store by reducing their bulk density. As compacted media do not show significant dust formation, it can also be concluded that they make for cleaner production suites, reducing contamination risk. The improved flowability also eases handling of the dry-powder media.
As well as powder properties, solubility behavior can also be optimized by means of compaction. Differences in solubility behavior are depicted in Figures 3A & 3B, which show powder and compacted Cellvento Feed-210, respectively, during dissolution in water. The powder is floating on the water surface even after 60 seconds of mixing, whereas the compacted formulation has already become suspended in the liquid after 16 seconds. This suspension behavior of the compacted formulation reduces overall dissolution time.
The solubility endpoint measurements, determined by turbidity measurement, revealed a threefold increase in dissolution rate, with the endpoint reduced from 22 to 7 minutes for this highly concentrated 81.60 g/L feed formulation.
The observed difference in solubility time between the powder and compacted formulation can be explained by the latter’s greater accessible surface. For the floating powder, the water-accessible surface corresponds to the diameter of the cylinder, whereas the compacted CCM formulation is surrounded entirely by the water, giving a much greater contact surface.
Even though the specific surface of compacted formulations is likely to be smaller than that of the powder, the accessible surface has a bigger effect on the solubility process. This can also be seen as an improvement in the CCM’s wettability. As such, compaction can significantly accelerate media preparation by increasing the dissolution rate of highly concentrated, slowly dissolving formulations, thereby removing a bottleneck from the media preparation process.
To prove the practical applicability of compacted CCM, scale-down fed-batch experiments were conducted. No impact on cell performance in terms of viable cell density or titer was observed (Figures 4A & 4B, respectively). No critical quality attributes, represented here by the example of the glycosylation pattern (Figure 4C), showed any alterations caused by compaction (data on charge variants, aggregation, and clipping not shown). Powder and compacted formulations perform identically in cell culture experiments. All of the above indicates compaction does not alter the properties of a CCM formulation.
The data summarized in this article demonstrate that compaction can enhance the efficiency of dry-powder CCM applications. Compaction can reduce shipping and storage costs by decreasing bulk volume. In addition, compaction can increase process safety by enhancing flowability and reducing dust formation. Finally, compaction can accelerate media preparation of slowly soluble formulations, thereby preventing this unit operation from becoming a production bottleneck. Crucially, these positive effects are not accompanied by impacts on cell-culture performance.
Nikolai Stankiewicz, Ph.D., is head of the technology transfer laboratory at Merck KGaA, Darmstadt, Germany. Website www.emdmillipore.com.