Benefits in using Microfiltration for enzyme cell separation

Microfiltration Illustration

Advances in biotechnology have revolutionized the commercial production of many biochemical products, especially industrial enzymes. The enzyme industry, both for commodity and specialty enzymes, is growing at a significant rate thereby creating pressure to improve the manufacturing efficiencies and economics of the harvesting and purification process steps used to produce the enzymes.

Typically manufactured using fermentation of genetically modified organisms, the traditional recovery methods in harvesting the cells and purifying the enzymes have been centrifugation followed by precoat filtration. Cross-flow membrane filtration, particularly microfiltration, has been increasingly used to replace either or both of these separation steps due to advantages in lower investment and operating costs, higher enzyme yields and simplification of downstream processing methods.

This paper will go into detail on how microfiltration can be used as the preferred cell harvesting step, outlining both the potential economic benefits as well as the process challenges encountered.

Enzymes

Industrial enzymes are potent effective compounds that can dramatically improve the speed or efficiency of biochemical reactions without altering the underlying process. Traditionally, use of industrial enzymes has been somewhat restricted because of a high sensitivity to surrounding conditions (pH, temperature, humidity, contaminants) and storage limitations. These disadvantages are being overcome with the development of recombinant enzymes that include vary specific, isolated complimentary DNA strands that enable them to be highly potent and efficient. Protein engineering, molecular evolution and other new protein design techniques are increasingly being used to further refine the characteristics and performance of enzymes.

Enzymes, sometimes referred to as biocatalysts, have great potential for improving reactions by increasing the effectiveness of, or even replacing, traditional chemical agents. Enzymes are often more economical than traditional chemicals, as well as being environmentally safer. As a result companies are investing substantial research and development effort in genetically modifying cells to produce highly focused enzymes. The most common uses today are for detergents (stain removal), textiles (wrinkle reduction), ethanol and leather, but potential applications probably number in the thousands.

Although advances in biotechnology have allowed engineering of enzymes for many applications, currently fewer than 30 enzymes account for more than 90% of the industrial use. This is expected to change as the number of niche and specialty enzymes increases, e.g., the use of phytase in animal feed. The market is growing at a healthy pace, with the broadened use of new enzymes making a significant contribution to this growth. Worldwide sales of industrial enzymes in 2003 were in excess of $2 billion.

Separation Technology

The development of new enzymes brings about the opportunity for new and improved recovery and separation processes. The science and technology now exists to produce these highly specific enzymes. One of the key challenges now is to refine and optimize the manufacturing of enzymes to make their production sufficiently economical to encourage growth in their use.

In genetic modification of cell DNA to either implant or modify certain characteristics, scientists make use of three main types of organisms: bacteria (pseudomonas is a common strain), yeast or fungal organisms as opposed to the mammalian cells used in the production of pharmaceutical products. All use fermentation technology to multiply the cells. The enzymes are typically, but not universally, extracellular, meaning they grow and are expressed outside the cells. Since the cell is intact, with the enzymes outside the cell, a physical separation is required to recover the enzymes from the fermentation broth. This is more straightforward than recovery of an intracellular compound, in which the cells must first be ruptured, creating a mixture of ingredients of multiple sizes and characteristics.

Microfiltration using ceramic membranes is quickly becoming the technology of choice for cell separation, holding back the whole cells while allowing the enzyme to pass through and be recovered in the permeate stream. It is an economically viable alternative to traditional separation techniques such as centrifugation and rotary vacuum filtration. Use of the more straightforward membrane filtration process to recover the cells allows biotechnology companies the ability to simplify their economics.

The conventional approach for primary cell separation in biotechnology has been either centrifugation or rotary drum vacuum filtration, followed by pressure leaf filters to remove the small amount of remaining cell debris. Once separated, the enzymes are concentrated and purified using ultrafiltration, a standard process that has been used in the industry for many years, before finishing and formulation.

Centrifugation has a number of drawbacks, such as high maintenance costs, heat production, protein denaturation, but most notably incomplete separation due to the fast centrifugation rely on density rather than size differential. Rotary drum vacuum filters and other dead-end filtration techniques also have disadvantages, most notably in terms of overall yield losses.

The modern approach is the use of cross-flow membrane filtration, and notably microfiltration. Several of the more important advantages of microfiltration over the conventional technologies are:

• It is generally more effective in purifying the enzyme, giving higher product yields while maintaining biological activity.
• It eliminates the pre-coat filter aids, such as diatomaceous earth (DE) or perlite that are used as filter media, doing away with the environmental issues associated with disposal of the used media. It also eliminates the cost and difficulties of purchasing and handling the filtration media.
• Cross-flow membrane filtration utilizes tangential flow, rather than dead-end filtration like a filter press. It can also be easily cleaned whenever necessary, using clean-in-place techniques and steam sterilized if required.
• It allows for better control of the process, and frequently improves the downstream ultrafiltration purification step.
• Perhaps most relevant to modern process economics, both the initial investment and ongoing maintenance costs are lower than with conventional centrifugation/dead-end filtration systems.

Figure 1 illustrates the replacement of a conventional separation process with microfiltration.

Figure 1 Typical Enzyme Cell Harvest / Recovery Process: Microfiltration replaces centrifugation and precoat filters

The implementation of microfiltration might differ depending on whether an enzyme producer is putting in a completely new process line or converting/upgrading an existing process. For a new system, it is more economical to use microfiltration to replace both the centrifugation and precoat filtering steps. Where upgrading an existing system, it may be desirable to just replace the precoat filters and leave the centrifuges in place, providing there are no maintenance issues with them.

Figure 2 Commercial Microfiltration Unit

Process and equipment design and pilot testing are all crucial in order to evaluate and define the specific operating conditions of a microfiltration separation process for enzymes to assure success on a commercial scale. The key is to design and run the microfiltration system properly to ensure getting the highest possible capacity to reduce capital costs, and the highest enzyme permeability, or passage of the enzymes through the membrane, to maximize yield and recovery. Figure 2 shows a typical commercial microfiltration system.

One of the biggest challenges in successfully designing a microfiltration system for enzyme recovery is achieving high permeability of the enzymes through the membrane. Controlling the boundary layer on the surface of the membrane is critical to ensuring good enzyme permeability. This has to be kept to an absolute minimum to make sure the membrane itself does the separation, and not the boundary layer. From a design standpoint, cross-flow velocity, which helps promote turbulence within the membrane channel and control of the proper trans-membrane pressure, is very important to accomplish this.

Key Membrane System Design Factors

To make the microfiltration system run efficiently, there are a number of different technical factors that tie in to impact the design and the overall performance of the microfiltration system.

Batch versus Continuous Process

Perhaps the first design factor to determine is the choice to run the system in either a batch or continuous mode of operation. A batch plant is often a more flexible solution than a continuous plant for cell harvesting, especially where the fermentation broth characteristics (viscosity in particular) are variable in nature. It is the recommended solution when processing small volumes such that the plant would be arranged in one or two recirculation loops and the amount of membrane area is not large.

When a larger broth volume is to be processed, requiring a configuration comprising five or more recirculation loops, then there are sufficient operating points to make a continuous plant more efficient, comparable in membrane area and therefore viable. The advantages of continuous operation include minimal residence time to minimize degradation, the ability to optimize each stage to match the anticipated viscosity, and minimize required membrane area. Diafiltration, which is the addition of water, buffer, alcohol or solvent to improve the recovery of specific compounds, can be automatically adjusted within each loop, and controls can be optimized accordingly.

There are, however, stringent demands on the control system in terms of maintaining a maximum pressure drop at the design recirculation rate if over-concentration is to be avoided. Microfiltration plants are typically designed to run with low operating pressure and a pressure drop of approximately 15 psi per element. A higher pressure drop may impair economics due to the higher energy cost associated with the bigger horsepower required, and possibly plant performance.

Choice of Membrane Type

Figure 3 Ceramic Membrane Elements and Housings

Experience has shown that ceramic membranes (Figure 3) are ideally suited for enzyme recovery. Due to the potentially high viscosity and cell density in processing whole cell fermentation broths, ceramic membranes are used because of the open, tubular channels (compared to the polymeric spirals, which have very narrow channels). The open-channel configuration, typically with 3, 4 or 6mm diameter channels, are easily able to process a whole cell broth without plugging by suspended solids. In addition, the ceramic membranes are sanitary, able to be cleaned in place or steam sterilized. They are FDA approved as a sanitary membrane they have been around for many years, and are commercially viable and technically capable.

When testing, the fundamental factor is choosing the right membrane to separate cell. Maximizing permeability will allow for high yield and recovery while minimizing the requirement for diafiltration water which has to be removed in the concentration step. The separation requires a membrane that has a pore size sufficiently large to allow the enzyme to pass through. The pore distribution might be important, as well, depending on the shape of the enzyme molecule. Other factors that impact the separation are surface charge of the membrane, convective forces, and electrostatic forces.

Surface Properties: The relative surface charge of the membrane itself compared to the enzyme is a prime consideration. If both have the same charge the enzyme may be repelled from the membrane surface, greatly reducing the transmission, whereas opposite charges will attract and potentially improve recovery. Membrane surface properties are also important from the point of view of fouling. A hydrophilic membrane is far more resistant to fouling than a hydrophobic membrane.

Convective Forces: Membrane filtration is a cross-flow technique, but, in fact, there are two forces at play: a shear force parallel to the membrane surface generated by the cross flow velocity, and a perpendicular force on the membrane surface generated by the trans-membrane pressure. It is this perpendicular force that is responsible for the formation of concentration polarization, or gel layer, a build-up of retained material on the membrane surface. It is important to run a microfiltration process with high surface velocity and low trans-membrane pressure, and the forces need to be optimized for proper operation.

Electrostatic Forces: The size and shape of the enzyme can change with pH and charge, changing the separation characteristics. This is particularly important with diafiltration, where the ionic strength such as salt concentration, can impact the permeability of the enzyme if there is a poor ionic balance. The presence of other proteins can also impact the membrane performance.

The Gel Layer

Formation of a concentration polarization, or gel layer, is a drawback of improperly designed ceramic microfiltration separation systems. Under pressure, the solvent and solute are forced against the membrane surface, resulting in an accumulation of rejected solute molecules, in this case the enzyme. This gel layer builds up on the membrane surface, acting as a secondary membrane and interfering with the separation.

There are two ways that formation of a gel layer adversely affects the separation. First, at moderate to high solute concentrations the resistance of the gel layer can be even greater than that of the membrane itself, effectively impeding recovery of the enzyme. Secondly, the enzyme that is caught in the gel layer is lost, reducing the overall yield.

In many cases the gel layer will tend to be denser when separating pre-clarified broth, which can contain small molecules and other proteins. Whole broth will be less affected and the larger cells may also cause a scouring effect which can help to minimize the gel layer. The gel layer is also affected by shear rate at the membrane surface.

Control of Gel Layer Formation

Ceramic membranes have the capacity to minimize the gel layer as long as the system is designed properly. By controlling convective forces such as (tangential velocity and trans-membrane pressure), the gel layer can be controlled and minimized. Typical velocity for this effect is 5 m/s or higher. This is the dramatic advantage for cross-flow membrane filtration over dead-end filtration, which exhibits only perpendicular forces on the filter media. An advantage of ceramic membranes is that if fouling does occur, they can be cleaned and put right back on line. Ceramic membranes also last for many years, before replacement is required, as opposed to filter cartridges, and other filter media, which are single use.

Fouling

Two types of fouling are encountered, reversible fouling where the flux increases and decreases with retentate pressure, and irreversible fouling, where the flux does not recover with a decrease in pressure. The flux must be maintained at a sustainable level, keeping fouling below a critical rate. The rate of fouling is related to the flux rate, and to control fouling there is a critical flux rate which must not be exceeded. This is particularly important during startup, where instantaneous fluxes can be high. Typically the MF plant needs to operate in the trans-membrane pressure range where flux increases proportionally with increasing pressure. At the point where the flux no longer increases proportionally, the critical flux has been exceeded and fouling can be irreversible without cleaning.

When designing the system, it is important to define the critical flux rate through pilot testing. In well-developed applications, experienced engineers have a pretty good idea what the critical flux rate will be. However, in new applications particularly within the biotechnology and pharmaceutical area, pilot testing is imperative. Process engineers focus on "excursions" in order to maximize capacity and minimize the gel layer, changing operating parameters such as temperatures, pressures and flow velocities until reaching the critical point. Once the optimum parameters are defined, those are then specified as the operating conditions for the commercial system.

Technical Advances in Ceramic Membranes

For many years companies have attempted to design membrane systems and products that will enhance system hydrodynamics by promoting turbulence in the boundary layer. Some of the early attempts included use of counter-rotating concentric cylinders or discs, introduction of gas bubbles, use of mechanical devices, and pulsed flow systems that reversed flow direction across the membrane. Many systems were tested on a laboratory scale but did not prove to be commercially viable.

Effective microfiltration systems have finally come on the market in the past few years that make it possible to control permeate pressure, and therefore average trans-membrane pressure, independently of tangential velocity. In particular, recent developments in ceramic membranes allow trans-membrane pressure to be controlled over the entire membrane surface, thereby achieving optimum sustainable product flux. Attempts to achieve this include: Two of the more prominent developments include these variable resistance in the membrane support layer and new variable resistance in the active membrane layer.

These advancements in the surface chemistry of the ceramic membranes allows for the modification of the gradient of the membrane in order to overcome the gel layer which can be critical in sensitive separation processes such as enzyme recovery.

Conventional Microfiltration Process (figure 4): Traditional ceramic membrane technology only allows control of trans-membrane pressure on a macro scale by individual adjustment of the feed and permeate pressures. The trans-membrane pressure along the length of any given element varies due to the pressure drop across the element. As ceramic elements tend to work at higher tangential velocities (up to 6 m/s) than many polymeric systems, there is significant variation in trans-membrane pressure, causing very fast formation of gel layer particularly at the inlet, thereby limiting transmission. The mid-point produces an optimum situation; while at the end of the element there is low flux due to lower than optimal trans-membrane pressure. As the feed travels along the membrane, less and less of the membrane area is actually utilized, so a gel layer forms very quickly, reducing the length of the membrane used efficiently.

Figure 4 Conventional Microfiltration Process

Various manufacturers have made modifications to the system design or the membranes themselves to produce "controlled gradient membranes" that reduce the gel layer, allowing for more difficult separations. This is done by controlling the gradient of the membrane, thereby producing a constant flux along the length of the entire element. This helps to maintain a consistent pressure drop over the whole element length thereby minimizing the gel polarization or boundary layer.

Advanced Ceramic Membranes: Two ceramic membrane manufacturers have come up with two different solutions for producing a controlled gradient membrane that minimizes the gel layer, allowing unique separations to occur. Both approaches modify the membrane gradient helps to accomplish the goal of eliminating or minimizing formation of a gel layer. One has a modified membrane support layer, and the other contains a variable-thickness active membrane layer.

The first approach (Figure 5) has a support layer with decreasing porosity down the length of the element in order to provide more resistance to flow at the inlet end and lower resistance at the outlet end. This helps achieve uniform permeate flux over the length of the membrane by allowing more flow through the support layer further down the element. This membrane is in use commercially and has proven to work in specific cases.

Figure 5 Variable Support Layer

The second approach (Figure 6) modifies the thickness of the membrane surface itself to accommodate the pressure drop across the membrane, being thicker at the inlet end of the element and thinner at the outlet. The decreasing membrane thickness reduces resistance down the length of the element to allow a constant flux across the whole length of the membrane. This membrane has also proven viable in commercial use ...

Figure 6 Variable Separation Layer

These two approaches in controlling the membrane gradient allow for a constant ratio of the pressure drop to membrane thickness, with the net result being a relatively constant permeate flux rate across the entire length of the element. With this constant flux rate, the boundary layer also remains consistent allowing for goal product permeability.

Conclusion

Controlled gradient membranes may not be required for each and every microfiltration system used to help in the recovery process of industrial enzymes. As with any significant process change, it is imperative to conduct lab and pilot scale tests with scaleable pilot plants to confirm the ability of ceramic membranes to effectively retain the cells and recover the enzymes.

There are also a number of other site specific factors that affect the economics of using microfiltration for enzymes. Nonetheless, microfiltration with ceramic membranes has proven to be an effective cell harvesting step that allows an enzyme produced to reduce processing costs and improving the overall economics of the enzyme manufacturing process.