Concentrating on Quality - Membrane Filtration (Continued)

Case Study 2 - Enzyme Production

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.

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 clone and isolate specific complementary DNA strands. Protein engineering, molecular evolution and other new protein design techniques are increasingly being used to 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 key is to refine and develop the manufacturing of enzymes to make 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 ingredients). All use fermentation technology to multiply the cells. The enzymes are typically but not universally extracellular, meaning they grow outside the cells. Since the cell is intact, with the enzymes expressed 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 widely used for this separation, holding back the whole cells while allowing the enzyme to pass through and be recovered in the permeate stream. It is an economically attractive alternative to traditional separation techniques such as centrifugation and rotary vacuum filtration.

The conventional approach for cell harvest has been use of a centrifuge or rotary drum filter to remove the bulk of the cells, followed by a pre-coat filter, or leaf press filter to purify the enzymes. Once separated, the enzymes are concentrated and purified using ultrafiltration before finishing (drying or other polishing) and formulation. Indeed this was one of the first applications for UF, dating back to the early 1970's, using plate and frame systems.

Centrifugation has a number of drawbacks, such as heat production, protein denaturation, but most notably incomplete separation centrifugation relying 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 resulting from the two stage separation.

The modern approach is the use of cross-flow microfiltration. It has several important advantages over the conventional technology:

• 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 diametaceous 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 utilises 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, due to the higher product purity.
• 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 4 illustrates the replacement of a conventional separation process with microfiltration.

The implementation of microfiltration might differ depending on whether a 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, thereby allowing a higher concentration factor within the MF plant and hence optimizing the volume of buffer solution required for a given yield, and hence reducing the demand on the ultrafiltration plant during subsequent concentration.

Unlike the dairy industry where the processes are well known, 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 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 5. A typical commercial microfiltration system.

Based on extensive experience in running long-term pilot plant programs, the biggest challenge in successfully designing a microfiltration system for enzymes 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, or gel layer.

In challenging separation applications such as enzyme processing, the high pressure drop across microfiltration membranes, resulting from an optimized cross flow velocity, can make it difficult to properly maintain the gel layer by having a potentially much higher flux rate at the inlet of the membrane due to the higher pressure. To overcome this, companies have developed a third generation of ceramic membranes with a defined membrane gradient producing a consistent ratio of pressure drop to membrane thickness, which has been shown to achieve a uniform permeate flux across the entire membrane length. This allows optimization to minimize the boundary layer and maximizing capacity and product permeability.

Hence with proper design of the membrane system using features such as controlled gradient membranes, microfiltration can be used as the preferred cell harvesting step to minimize the processing costs and maximize the economics of the enzyme manufacturing process.

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