"The fate of leachate" in upstream and downstream processes of biopharmaceuticals

Dr. Armin Hauk; Ina Pahl; Roberto Menzel; Samuel Dorey; Dr. Isabelle Uettwiller
(The above authors are all from Sartorius Steadi biotechnology)
Translation Review: Sun Yilin, Shen Liang (Sedolis Validation Service Team)
The single use system (SUS) is widely used in biopharmaceutical production. Compounds in these polymeric materials may enter the manufacturing process as leachables at different stages, may affect production process efficiencies (such as inhibiting cell growth) or become process-related drug impurities, potentially affecting drug quality and/or patients. Safety.

To better understand the range and concentration of leachables in dynamic processes and even in finished formulations, we propose a paradigm shift for the concept of "the fate of leachate". Based on the physico-chemical mechanism, the "destiny of the leachate" is described by source, distribution, and descent.

The source of the leachate under dynamic process conditions can be described in terms of Fick's second diffusion law and polymer-matrix interfacial mass transfer ("flux"). Based on this model, the kinetics and final equilibrium concentration of the leachate can be predicted.

In any biopharmaceutical process, the adsorption and elution processes significantly affect the leachate levels, such as downstream filtration, separation and purification steps. In view of the reversibility of adsorption, especially those process steps for removing the adsorbate, it can be used as the "end point of disappearance" of the leachate.

It is conceivable that the leachate of the disposable bioreactor bag is adsorbed on the surface of the host cell and on the cell debris. Removal of host cells and cell debris during the harvesting step also removes the adsorbed leachate, and only the truly dissolved extract remains in the process.

Previously we were able to demonstrate that the filtration membranes in the filtration and membrane adsorption equipment were able to remove the leachate very efficiently during the purification step. The ability to remove typical materials is measured in μg/cm 2 membrane surface area or μg/cm 3 membrane adsorption bed volume.

Magarian et al. introduced a study using UF/DF to remove leachables. They demonstrated that the leachate was removed by infiltration and conformed to the traditional UF/DF mechanism. In addition, more process steps in the production process can reduce the leachate content. The elution solution removes the leachate during ion exchange or affinity chromatography. For adsorption components, chromatographic separation is also a way of leaching.

In order to achieve the computability of the source, distribution and decline of the leachate, it is only necessary to consider them comprehensively and to calculate the "destiny of the leachate" in each process step and throughout the downstream production. Figure 1 shows a dynamic unit model for calculating the leachate content of a hypothetical process. The model treats process steps and/or process equipment as separate units that are combined to simulate the entire process. In each unit, the source, distribution, and descent of the leachate are calculated based on the basic physico-chemical mechanism. Using material balance conditions, the exchange or discharge between units is simulated by liquid phase flow.


Figure 1 assumes a dynamic unit model for subsequent process steps in downstream production of biopharmaceuticals
The input data process unit comprising: the total volume of the solution, the total mass of the polymer phase, the polymer thickness and surface area, the amount of the original polymer m o extract, biomass disposable bioreactor bag, sorbent Mass and / or surface area, as well as the dialysis volume in the UF / DF step. The input data required for the leachate include: the diffusion constant of the polymer (D), the partition coefficient between the polymer and the solution (K P/L ), the partition coefficient between the biomass and the solution (K D-bio ), filtration The specific capacity of the device and the purification device (Kap filter ) and the UF/DF factor (z).
Table 1 shows the results of the unit model calculations for the two hypothetical compounds A and B in Figure 1. The physico-chemical parameters of the Leachate A (Table 1) can be described as similar to the degradation products of the additive such as the di-tert-butylphenol isomer, and the Leachate B is similar to caprolactam. For both compounds, we assume that they are present in the raw material (leached from the packaging container) and migrated from the contact material during the preparation phase of the medium (mixed and stored for 24 h). For extracts A and B, it was observed that the concentration of the medium in the disposable bioreactor bag increased within 21 days (the red line is shown); during this period, the leachate A from the disposable bioreactor bag polymer material migrated The amount of B and B gradually decreases (the green line is shown). Since the leachate A has a tendency to be adsorbed by biomass, it can be removed from the process by separating the cell debris (blue line shown). This effect is significantly weaker for Leachate B because of its good water solubility and a lower propensity for biomass adsorption. The amount of both extracts in the process solution increased during the subsequent treatment of the process solution and the 24 h storage phase. The filtration process is capable of removing a portion of the extracts A and B through the filter.
The following UF/DF steps are used to remove Leachate B very efficiently and are more efficient than Leach A. Leachate A has a lower z value and therefore a higher tendency to remain in the reflux end, while leachate B has a z value of 0.7 and is more effectively removed from the process by permeation. For leachates A and B, although already present in the raw materials and constantly migrating out of the contact material during the production process, the combination of different downstream process steps can significantly reduce the level of leachate in the final product. In summary, the purpose of downstream production is to “purify” the product, effectively removing the possible leachables as well as other unwanted process impurities. In addition, the calculations show that the concept of “near” patients is reasonable as a common risk assessment method and can be supported by our model calculations.

Table 1 : Summary of input parameters for extracts A and B ; calculated amounts of A and B in different units and final content of A and B in formulation ( DP )
Input parameters of leachate A :
D = 2,0E-10 cm 2 /s K P/L = 1000
K D-bio = 100 Kap filter = 5 μg/cm 2
UF/DF factor, z = 0,5
Input parameters of leachate B :
D = 8,0E-10 cm 8 /s K P/L = 1
K D-bio = 5 Kap filter = 5 μg/cm 2
UF/DF factor; z = 0,7
Yield of extract A; predicted amount in finished preparation: 6 mg
Yield of extract B; predicted amount in finished preparation: 28 mg
Although the method for predicting the leachate in the overall process is based on mathematical model calculations, the results reflect that the actual leachate condition is significantly better than the "worst condition". Model calculations show that, despite the multiple sources of leachables, the decline in leachables in biopharmaceutical processes is important and needs to be considered in the assessment of leachate (risk).

The Sartorius technical team has begun to further research and refine the model unit to include possible leachate reactions, especially potential leachate-protein interactions. After the model has been optimized and fine-tuned, it needs to be further verified by comparing the model results with the actual measured values ​​of the pilot or production scale.

The Sartorius Validation Services team is a pioneer in extractables and leachate research and has provided more than 20 years of analytical testing and regulatory technical support. Sartorius provides the most accurate interpretation of current regulatory requirements and industry standards, collaborates with customers to meet regulatory expectations, and identifies appropriate extractables and leachate designs for their pharmaceutical formulations based on the customer's actual process conditions.
references
Hauk A., Pahl I, Menzel R., Dorey S and Uettwiller I.: On the Fate of Leachables: An Introduction of a Concept to Investigate Leachables with a “Holistic” or System Approach; ECI Conference, Tomar Portugal, 8th -10th May.2017
Hauk A.: On the “Fate of Leachables” in Biopharmaceutical Up-Stream and Down-Stream Processes; Vonlanthen E&L-Summit, 19th October 2017, Berlin
Magarian N., Lee K., Nagpal K., Skidmore K. and Mahajan E.: Clearance of Extractables and Leachables from Single Use technology via Ultrafiltration/Diafiltration; AIChE Publication, 2016
About Sartorius Steadi
Sartorius Stedim Biotech is a leading international provider of equipment and services for the biopharmaceutical industry, providing a safe, timely and cost-effective integrated solution for the development and production of biopharmaceuticals worldwide. As a complete solution provider, Sartorius Steyr offers a portfolio of products covering almost all steps of the biopharmaceutical process. The company is committed to promoting single-use technology and value-added services to meet the rapidly growing technology needs of the biopharmaceutical industry. Headquartered in Aubagne, France, the company is listed on the European exchange in Paris; it is known worldwide for its production and R&D centers in Europe, North America and Asia, as well as its worldwide sales network.
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