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The viscosity of oily emulsions can reduce filter capacity and bacterial retention.
Adjuvants, pharmacological or immunological agents that increase the immune system's response to the antigen in a vaccine, are becoming more common in vaccine formulations. Oil-in-water emulsions and liposome adjuvants in particular are being pursued by vaccine manufacturers as a cost effective way to meet worldwide vaccine demand. More robust filtration solutions are needed to achieve sterile filtration of these streams, since they can be difficult to validate for desired capacity and retention under desired process conditions. A team of researchers conducted an investigation of the variables that affect capacity and bacterial retention during filtration of a model emulsion adjuvant. Based on the results, we developed and manipulated the model stream to determine how to optimize the process for improved performance. The study found that processing conditions, filter selection, and feed stream properties all affect filtration performance. Temperature, pressure, membrane selection, and particle size and loading can be manipulated to improve process efficiency and ensure product sterility.
Adjuvants increase the response of the immune system to the antigen in the vaccine, improving its efficacy and enabling reduced dosage to achieve the desired immune response. In use since the 1930s, the oldest adjuvant is alum (aluminum salts), which is still used in the industry today. Since aluminum salts are >0.2 μm, they cannot be sterilized by 0.2 μm filtration, so typically they are heat sterilized instead.
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Newer adjuvants, including oil-in-water emulsions and liposomes, were introduced to the market more than a decade ago. These novel adjuvant formulations are more universal than alum and are thought to minimize adverse patient reactions. They are approved for use in vaccine formulations in Europe, Asia, Canada, and Mexico. The benefits of these novel adjuvants are driving their increased use for the foreseeable future.1,2 This increased use has brought more attention to the validation and filtration challenges associated with each. Millipore undertook a study to better understand the mechanisms that affect capacity and bacteria retention in the presence of emulsions and liposomes, and use this understanding to develop robust filtration solutions.
Using an oil-in-water emulsion as our model stream, the study team investigated the mechanisms that affect capacity and retention. Throughput of a membrane in a typical filtration application is affected by many factors, including membrane structure, the viscosity of the suspension, particle size, particle concentration, and filter train resistance. The filter's overall throughput is determined by flux and capacity. Flux is determined by driving forces (e.g., inlet pressure), stream properties (viscosity), and the membrane structure (i.e., pore size, asymmetry). Capacity is also driven by the membrane structure and by properties of the process stream, such as particle load.
Figure 1. Relative capacity of a symmetric and an asymmetric membrane for a model oil-in-water emulsion. The asymmetric membrane has a much higher capacity in this stream, which can be attributed to higher intial flux.
The impact of membrane structure on capacity was demonstrated by comparing two membrane structures that varied in asymmetry. As Figure 1 shows, the more asymmetric structure had a higher capacity, which we believe was associated with higher initial flux. Regardless of membrane structure, the flow rate was very low, even early in the filtration process. The viscosity of the model emulsion was one of the factors that contributed to the observed throughput. As Figure 2 shows, the viscosity of the emulsion is high compared to that of water. As a result, the flux of the emulsion is lower than the flux of a typical aqueous stream. The reduced flux significantly affects processing time and filter sizing.
Figure 2. Viscosity of the model stream as a function of temperature. As expected, the suspension is less viscous at higher temperatures, but at all temperatures, its viscosity is higher than that of water, which is 0.01 poise at room temperature.
Testing was done to observe the plugging behavior of membranes when filtering the emulsion. In addition to the low initial flux, the flow declined rapidly after the filtration started, suggesting that the membranes were plugging quickly. To investigate the impact of the model stream on membrane plugging, we manipulated the size and concentration of particles in the model stream. Smaller particles increased capacity, as shown in Figure 3. This is an indication that particle plugging is also an important factor in filter capacity.
Figure 3. Relative capacity increases as average particle size decreases.
To confirm that pore blockage was the mechanism of membrane plugging, we used common fouling models to fit our data. Fouling models use approximations of how a filter plugs to fit the fouling curves (volume versus time). Typical fouling models include caking (the build-up of particles on the filter surface), complete pore blockage (blocking of a pore opening by a single particle), and gradual pore plugging (build up of particles within a pore to gradually restrict flow).3 By plotting our data using the standard fouling models, we found that the classic Vmax model,4,5 which is based on gradual pore plugging, best fit the data, and accurately predicted the plugging performance and overall capacity. This analysis provided further evidence that pore blockage was the primary mechanism of flow decay.
Although our testing showed that the classic Vmax model predicted the flow decay behavior, we also found that the plugging behavior was pressure-dependent. Capacity increased with increasing pressure drop across the membrane, in some cases disproportionately to the increase in pressure. Figure 4 shows the impact of pressure on filter capacity, which was non-linear. A number of factors may contribute to this behavior, one of which is suspected to be the high viscosity of the emulsion compared to a typical aqueous stream. Several follow-up experiments confirmed this theory.
Figure 4. Dependence of capacity on pressure. Capacity increases with increasing pressure. The same membrane was challenged, in duplicate, at constant pressures of 5, 7.5, 10, 15, 20, and 30 psi. Capacity was determined as the volume at which the flow had declined to 10% of its initial value in the emulsion.
Sterilizing-grade filters are used to ensure product sterility and are designed to remove all bacteria present in a process stream. Regulatory requirements dictate that processes involving sterile filtration must be validated to remove at least 107 bacteria per square centimeter of filter area under worst-case processing conditions. ASTM F838-83 outlines the standard method for determining the bacteria retention of a membrane using Brevundimonas diminuta (ATCC 19146).6 Using our model stream, we investigated mechanisms that affect bacteria retention; our testing followed this ASTM test method. In our study, two primary factors influenced retention: the suspected interaction among the oil emulsion, the bacteria, and the membrane, and the impact of plugging.
The coating of bacteria on the membrane with emulsion is likely one of the contributing factors that makes bacterial retention less robust in the presence of the emulsions than in typical aqueous solutions. Scanning electron microscope (SEM) imaging was used to evaluate bacteria size. Our analysis did not show bacteria to decrease in size on exposure to the oil emulsion; however, the bacteria did appear to be coated with emulsion. Additional studies showed that the emulsion did not affect bacteria viability. Other properties, such as motility and flexibility, have not been evaluated yet in detail, but to date, no evidence has been observed to support the suggestion that bacterial retention is affected by changes in their properties.
Pore blockage, the primary flow decay mechanism, also affects retention. Similar to the phenomenon observed in virus filtration, retention in the presence of the emulsion decreases as the membrane plugs. This behavior is exacerbated by the presence of the oily emulsion coating the bacteria, which produces a worst-case environment for retention, even in the absence of membrane plugging.
The study also assessed the effect of temperature on retention and found better retention at higher temperatures—approximately 1 log higher at room temperature than at cold temperatures. We are conducting further investigation to determine the reasons behind this finding.
In our study, using a model oil-in-water emulsion, we found that capacity limitations are primarily the result of pore blockage, coupled with low initial flux. Additionally, process conditions, such as inlet pressure, can further reduce membrane capacity. These factors also affect bacteria retention and are exaggerated by temperature effects and the interaction of the oil and the bacteria.
Based on improved understanding of the mechanisms involved in filter capacity and retention in the presence of an emulsion, there are a number of variables that can be manipulated to optimize process performance. Processing conditions, filter selection, and feed stream properties affect on filtration performance. Temperature, pressure, membrane selection particle size, and loading can all be manipulated to improve process efficiency and ensure product sterility. Combinations of the various options present the potential for further improvements.
Early process development work coupled with early prescreening before validation is recommended to ensure a robust process under worst-case conditions for temperature, pressure, and particle load.
Christina Carbrello is a development engineer and Marc Rogers is a senior microbiological scientist, both at Millipore Corporation Billerica, MA, firstname.lastname@example.org, 781.533.2141.
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