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Introduction

The speed with which homogeneity is reached within solutions is a critical performance attribute of single-use mixing systems. These systems are commonly used in biopharmaceutical and pharmaceutical media and buffer preparation processes, often involving the hydration of a powder. Recirculation mixing performed using a peristaltic pump can provide a simple and cost effective alternative to more elaborate mixing systems such as those which incorporate a magnetically driven impeller.

This technical bulletin highlights the results of conductivity testing to characterize recirculation mixing performance of single-use systems deployed in Meissner’s QuaDrum^® rigid outer containers (ROCs). All three standard QuaDrum^® ROCs, with nominally rated volumes of 50 L, 100 L and 200 L, were evaluated with corresponding single-use systems. The scope of this testing included varying flow rates and mixing directions while using NaCl as the mixing solute.

Materials and Methods

Electronic conductivity meters (Mettler Toledo InPro 7108-TC-VP and InPro 7100) were mounted and sealed into locations which allowed for data capture at the top, middle and bottom of 50 L, 100 L and 200 L biocontainers deployed in QuaDrum^® ROCs (as

shown by the drawing in Figure 1). The QuaDrum^® ROCs used for this testing included the optional bottom drain version (part numbers FASD-050B, FASD-100B and FASD-200B) which was installed on their corresponding accessory dollies to facilitate the use of a bottom mounted fluid path. A volume of water equivalent to the nominally rated capacity of each (i.e. 50 L, 100 L or 200 L) was added to each single-use assembly. The recirculation flow was established using a Masterflex^® I/P peristaltic pump set to the desired rate that was verified via an offline measurement. Mixing direction was controlled based on the direction of recirculation flow through the assembly and was either top to bottom (i.e. fluid evacuated from the top of the closed system and returned through the bottom port) or bottom to top (i.e. fluid evacuated from the bottom of the closed system and returned through the top port). NaCl (VWR GR ACS Sodium Chloride) was introduced into the system through a large bore 3″ TC top port in the quantity necessary to achieve a concentration of 15 g/L for the given fluid volume. The system was allowed to mix until all three of the conductivity readings were stable. Five tests were conducted, which included analysis of all three fluid volumes with varying flow rates and mixing directions. The specific test conditions are presented in Table 1.

Results and Discussions

The conductivity testing results are presented in Table 1 and Figure 2. The response from the bottom sensor displayed a characteristic overshoot before settling to equilibrium for each of the test conditions. This can be explained due to initial NaCl accumulation near the bottom of the biocontainer immediately following solute addition. The middle level sensor also followed a similar response to that of the bottom sensor but with a smaller overshoot. The top sensor displayed a varied response based on the specific test being performed. During tests 1 and 4 the top sensor gradually approached the equilibrium conductivity value while during tests 2 and 5 it displayed a delayed response with a few spikes in conductivity. In Test 1 (Figure 2A), all three sensors reached equilibrium conductivity at about 75 seconds and follow the general patterns described above. Test 2 (Figure 2B), showed an unusual response from the top sensor, which spiked around 35, 47, and 63 seconds.

This is likely due to a relatively low flow rate for the larger liquid volume with a time to equilibrium of 105 seconds. Tests 3 and 4 (Figures 2C and 2D) examine the effect of recirculation flow direction on a 100 L single-use assembly deployed in a QuaDrum^® ROC. These tests were performed using a pump-Y element, as opposed to a single lumen pump tubing segment, in order to achieve an increased flow rate of 17 LPM. The difference in performance is significant as it took 45 seconds to reach equilibrium for top to bottom recirculation, whereas bottom to top recirculation took 85 seconds. In Test 5 (Figure 2E), all three sensors reached equilibrium at around 105 seconds. The response of Test 5 appears similar to that shown in Test 2, with an overshoot at the bottom and middle with a delayed response at the top sensor. This is explained by the bottom becoming heavily concentrated while the top experienced mixed concentration levels. At a flow rate of 17 LPM, the 200 L mixing assembly takes a few minutes of mixing to reach a consistent concentration level.

Conclusion

The results of conductivity testing using QuaDrum^® ROC based single-use mixing assemblies indicate that achieving an evenly mixed solution within a relatively quick period of time is possible using this technique. The testing shows the significance of using an appropriate flow rate predicated on the overall volume of the system. It further demonstrates that for powdered solutes with a specific gravity greater than one, employing a mixing direction that circulates fluid from the top of the system and returns it to the base, is preferable. Therefore, under the appropriate operating conditions, recirculation mixing using QuaDrum^® ROCs with installed single-use biocontainer assemblies can provide an effective means to achieve and maintain homogenous solutions.

For more information or test data, please contact Meissner Filtration Products.

Recirculation Flow Testing a FlexGro^® Biocontainer Fitted with an Anchored Dip Tube for Perfusion Culture

Introduction

Perfusion processes are rapidly being adopted as an alternative to traditional batch and fed batch culturing of mammalian cells. In one type of perfusion system, a bioreactor is connected to a recirculation pathway where the exchange of spent media occurs. Rocker bioreactors, also commonly referred to as wave bioreactors, have been a popular choice for seed train applications and small scale protein production for required working volumes below the 100 L range. The continuous rocking motion of the reactor generates a wave inside of a single-use biocontainer, allowing aeration of the cell culture while avoiding problematic high shear conditions. However, one commonly encountered challenge of using rocker bioreactors in perfusion mode is the undesirable transfer of air into the recirculation pathway. These entrapped air bubbles compromise the effectiveness of the perfusion filter, which is usually accomplished via either a tangential flow filtration (TFF) or an alternating tangential flow filtration (ATF) device. This technical bulletin evaluates a solution to enable perfusion culture via a new anchored dip tube that was designed to prevent air migration into the recirculation pathway. A 50 L FlexGro^® biocontainer connected to a recirculation loop was tested for the presence of entrapped air under varying processing conditions of liquid volume, rocking rate, and rocking angle. The experimental data provides insights into the relationship between liquid volume, rocking rate, and rocking angle, which is useful in defining the proven acceptable range (PAR) of processing parameters for the perfusion system.

Materials and Methods

A Meissner 50 L FlexGro^® biocontainer (part number B12R00505-006) modified to incorporate a ¼” x 7/16″ (ID x OD) anchored dip tube was mounted to a rocker bioreactor (GEHC, Wave Bioreactor 20/50 system), filled with dyed water, and inflated with air to approximately 1 psi, according to the manufacturers instructions. A recirculation flow pathway comprised of transparent silicone tubing was then established by interconnecting the inlet and outlet tubing. A peristaltic pump (Masterflex^® I/P) was used to maintain a constant recirculation flow rate of 8 L/min. The testing setup is shown in Figure 1. Rocking angles of 6-10°, rocking rates of 15-35 rpm, and liquid volumes of 10-25 L were used in combination to determine whether air transfer into the recirculation pathway occurred. Table 1 summarizes the testing parameters. The recirculation pathway was visually inspected for migration of air bubbles for each of the operational combinations tested.

Results and Discussions

The graph presented in Figure 2 shows the maximum rocking rate determined as a function of rocking angle and working volume before air migration occurred. A recirculation flow rate of 8 L/min was used in order to represent worst case conditions and amplify the migration of air through the tubing. As hypothesized, the rocking angle, rocking rate, and working volume all had an effect on the transfer of air into the tubing. An increase in rocking angle and rocking rate contributed to increased air transfer, while a decrease in liquid volume also led to an increase in air transfer. To prevent air migration, meeting two conditions seemed to be crucial. First, the anchored dip tube must remain submerged below the liquid level at all times. Second, the liquid near the dip tube cannot contain air bubbles. All three operating parameters of volume, rocking angle, and rocking rate contributed to whether the first condition was met. Although the anchored dip tube was positioned along the centerline of the biocontainer, air entrapment still occurred under certain conditions, even when the highest working volume of 25 L was used. When the rocking rate was increased high enough (above 30 rpm for an angle of 8° or greater) the inertial forces generated caused the bulk of the wave to move beyond the centerline, resulting in a liquid level below the anchored dip tube inlet opening. The primary contributor to whether the second condition is met is the rocking rate. At higher rocking rates, increased agitation resulted in turbulent flow patterns, which produced air bubbles in the liquid. It is likely that this effect will be further exacerbated during actual cell culture due to the generation of foam. It is expected that the 50 L biocontainer used is representative of a worst case condition when compared to the smaller 20 L and 10 L FlexGro^® biocontainers because the effect of the inertial forces in generating turbulent mixing and agitation is expected to decrease at lower volumes.

Conclusion

A 50 L FlexGro^® biocontainer modified with a centerline dip tube to effect a recirculation loop supports its use in perfusion applications, provided that the operational culture conditions used remain within the PAR to avoid air entrapment. Further experimental evaluation may be required using actual cell culture conditions in order to define a normal operating range (NOR) of operational parameters suitable for perfusion culture. Therefore, the results presented in this technical bulletin should only serve as an initial guide towards the adoption of FlexGro^® biocontainers for perfusion culture.

For more information or test data, please contact Meissner Filtration Products.

Flexgro Recirculation Flow Testing

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Pharmaceutical Technology: Innovation Outlook – Scale-up by Scaling Out

Industry standard capacity implementation typically sees a progression from process development (PD) to pilot plant to scale-up or full scale production. While this standard is not going away anytime soon as conventional large-scale production facilities continue to be planned, and PD/pilot activities are certainly not being obsoleted, traditional scale-up is being challenged by scale-out, and the accepted norm of full scale production is shifting. Conventional large scale facilities certainly have their place in biopharmaceutical manufacturing. However, a shift toward flexible manufacturing capacity is an industry trend that provides a litany of benefits for tomorrow’s needs. These include rapid pandemic response, planning for variable production capacity of products predicated on dynamic market demand driven by competition of biosimilars, manufacturing closer to affected populations bases, and ultimately producing drugs for smaller numbers of patients as treatments become more tailored to specific conditions. To accommodate these needs, given advancements in titers from fed-batch processes and the advent of continuous processing, scaling-out via simply replicating a given process at a fixed volume, is often a better and more efficient solution relative to scaling-up.

The move towards treating ever smaller affected population bases, via treatments more specifically tailored to a given ailment, ultimately leads to personalized medicine. The manufacture of these therapies, despite processing commonalities, by nature, requires scaled-out production via replicating a similar process, at a given volume, to a significant degree. Scaling-out benefits from (and to some degree is predicated upon) the employment of single-use systems (SUS). These systems are not new; however, when applied in the context of scaling-out, wherein larger quantities of relatively smaller SUS may be consumed in increasingly complex and critical applications, the associated common challenges can become exacerbated. These challenges include the ability to rapidly and repeatedly deploy SUS, logistics considerations, and the ability to establish a robust supply chain to ensure consistent delivery of product that meets stringent quality specifications. Further, it is critical that suitable products exist in form factors commensurate with the process scale. What may have historically been a small laboratory filter may now be a critical final filter, and therefore needs to be supported in the same manner as its larger, traditional process scale, counterparts. Meissner is addressing this via new small-scale filtration products designed specifically to meet these needs.

Scaled-out processes also benefit from, and often require, enhanced levels of automation. For drug manufacturers, this is predicated by running a greater number of processes, typically concurrently, and perhaps in advanced operational modes. Likewise, the need for increased automation exists within the supply base which supports scaled-out processes, both in terms of providing integrated process solutions, as well as advancements pertaining to consumables manufacturing methods and technology. Common automation drivers for both drug manufacturers and the supply base include achieving higher degrees of process robustness, rapid augmentation of capacity, and the ability to codify additional manufacturing data. Meissner is addressing this by developing highly automated unit processing solutions as well as next generation SUS connection technology that replaces manually assembled mechanical connections with permanent thermal bonds generated via wholly automated processes.

GEN Roundup: Filters’ Future Focuses on Functionality

This Genetic Engineering & Biotechnology News (GEN) article delivers input from bioprocess purification specialists involved in downstream biopharmaceutical production, as they discuss key advances in virus filtration over the last 10 years, with input on what is necessary for successful and economic large-scale filtration. GEN’s expert panel reviews filtration’s triumphs, tradeoffs, and future trials to come.

The panel includes Leesa McBurnie, Meissner Filtration Products’ Manager of Laboratory and Validation Services, as well as input from GE Healthcare Life Sciences, MilliporeSigma, Pall Life Sciences, Sartorius Stedim Biotech, and Spectrum Laboratories.

INTERPHEX New York 2016 Video covers Meissner Filtration Products’ single-use systems and filtration systems innovations to optimize biopharmaceutical manufacturing.

Watch and learn about Meissner’s latest innovations for optimized manufacturing in the biotech and pharmaceutical industry. Max Blomberg, Director of Operations, introduces BioLink^® Modular Overmolding – a disruptive technology that unifies all of the benefits of traditional overmolding with the speed and flexability of traditional mechanical connections. Optimized for single-use applications, BioLink^® assemblies allow Meissner’s Applications Engineering Team to create single-use assemblies tailored to the specific requirements of the end-user that result in reduced process risk.

Christian Julien, Director of Pharma Process Solutions discusses the FluoroFlex^® PVDF biocontainer for applications beyond the limits of traditional polyolefin-based biocontainers. Optimized for the pharmaceutical industry’s most demanding requirements, FluoroFlex^® biocontainers open new opportunities for single-use systems. Antioxidant and additive packages are not needed in the manufacture of this film, which makes it an ultraclean alternative to polyolefin films, with an extremely low leachables and extractables profile. The FluoroFlex^® biocontainer’s excellent performance in cell culture applications due to the lack of antioxidants is highlighted.

American Pharmaceutical Review March 2019 Single-Use Roundtable

This American Pharmaceutical Review article discusses the best practices for implementing single-use (disposable) technologies, and the consequences of understanding that as an end-user, you are effectively outsourcing functions were serviced via in-house capacity with traditional systems. The easiest processes to transition to single-use systems are highlighted with a focus on implementing best practices for integrating single-use and stainless steel equipment (hybrid systems). Predictions for the future of single-use systems are discussed. The panel includes Max Blomberg, Director of Operations, and Christian Julien, Director of Pharma Process Solutions, as well as experts from Finesse, BioPlan Associates, Pall Life Sciences, Thermo Fisher Scientific and Ulteemit Bioconsulting.