Problem 11 - Bioseparations Science and Engineering

Question

Stevia Sweetener Stevia is a leafy green plant native to subtropical and tropical regions (http://en.wikipedia.org/wiki/Stevia). It is grown around the world for its sweet leaves, which are used in a variety of forms as a high- intensity sweetener. Stevia extracts have \(200-300\) times the sweetness of sucrose and do not raise blood glucose. Stevia-based sweeteners are used today in dairy products, health drinks and carbonated beverages. Both Coca Cola and PepsiCo have introduced drinks containing stevia-based sweeteners under the commercial names of Truvia and PureVia, respectively. Stevia sweeteners are presently extracted from the leaves of stevia plants. However, recent advances in synthetic biology have enabled the production of stevia sweeteners via fermentation. Based on information from the technical and patent literature, design and evaluate a fermentation process for producing \(5,000,000 \mathrm{~kg}\) of stevia sweeteners per year for applications in the beverage industries. Assume that production is accomplished via fermentation in yeast that secretes stevia molecules, reaching a product titer in the fermentation broth of \(75 \mathrm{~g} / \mathrm{liter}\). The purification train includes a disk-stack centrifuge for biomass removal and a sequence of chromatography, membrane filtration, crystallization, and drying units for the isolation and purification of the product. Your analysis should include overall material and energy balances, equipment sizing, and estimation of capital and operating cost. Furthermore, estimate the profitability of the investment assuming a selling price for the final product equal to 200 times the current price of sucrose. 12 MAb Production in Stirred Tank Bioreactors with Disposable Bags The MAb example in section \(12.6 .3\) analyzes a process for producing \(1,544 \mathrm{~kg}\) of purified MAb per year using four 20,000 liter stainless steel production bioreactors operating in staggered mode (out of phase) and feeding a single purification train. The product titer is 2 \(\mathrm{g} / \mathrm{liter}\) and the cycle time of each bioreactor is 2 weeks. In the last few years, new cell lines have become available (e.g., PER.C6 from Percivia) that can reach significantly higher product titers ( > \(20 \mathrm{~g} / \mathrm{liter}\) ). Deployment of such cell lines greatly reduces the volume of the upstream equipment and enables single-use systems to produce metric ton quantities of MAbs. Rocking and stirred tank bioreactors that utilize single- use (disposable) liners (bags) have become popular in the biopharmaceutical industry because they eliminate the need for cleaning and sterilization-in- place. Other advantages of such systems include increased processing flexibility and shorter validation, start-up, and commercialization times. Single-use bioreactors are available with working volume of up to 2,000 liters. Design a process using the new technologies described above which can produce \(1,200 \mathrm{~kg}\) of a therapeutic monoclonal antibody per year. Assume you make use of the PER.C6 cell line that can consistently reach \(10 \mathrm{~g} / \mathrm{liter}\) of product titer. For product purification, assume that you need two adsorptive chromatography steps (e.g., affinity and hydrophobic interaction) followed by a polishing ion exchange membrane adsorber that operates in flow-through mode (the product flows through the unit but certain DNA and other charged impurity molecules are retained by the membrane). Your analysis should include overall material and energy balances, equipment sizing, and estimation of capital and operating costs.

Step-by-Step Solution

Answer

The short answer would carry the profitability estimates derived for both problems given above. Each profitability would be derived from subtracting the total cost from the predicted revenues for each separate production process.

Step 1: Problem 1: Design and Evaluation of Stevia Sweeteners Production

1. Draw the block diagram of the process where yeast is used for fermentation. \n2. Calculate material balance around each unit. \n3. Perform an energy balance around each unit operation. \n4. Estimate the size of equipment required (fermentation tank, centrifuge, and other units) given the production specifications. \n5. Estimate the capital and operational cost considering the equipment price, operation & maintenance costs and the cost of raw materials. \n6. Compute the predicted revenue considering the selling price [use 200 times the current price of sucrose]. \n7. Calculate the profit by subtracting the total cost from the revenue.

Step 2: Problem 2: Designing a Process for Monoclonal Antibody Production

1. Sketch the process diagram considering the process parameters given in the question. \n2. Perform material balance around each unit operation, considering the properties of the new cell line. \n3. Perform an energy balance around each unit. \n4. Estimate the size of single-use bioreactors and other equipment required for the process, considering the production requirements. \n5. Estimate the operational and capital cost considering the equipment cost, operation & maintenance costs and raw materials. \n6. Estimate the profitability using the costs and potential revenue.

Key Concepts

Fermentation Process Design

When designing a fermentation process, it's crucial to visualize the entire workflow. Imagine yeast as tiny biological factories producing stevia sweeteners. The process typically starts with preparing the fermentation medium, which acts as the growth environment for yeast. Proper medium composition is essential to maximize yield and productivity.

In our example, yeast is selected for its ability to secrete stevia molecules into the broth. Ensuring the right temperature, pH, and nutrient supply optimizes yeast activity and product concentration. This is where our target titer of 75 g/L comes into play, meaning we aim for 75 grams of product per liter of fermentation broth. The process flows into downstream operations, beginning with a centrifuge to separate biomass from the liquid.

Each step, including chromatography and drying, needs precision to maintain product quality. The design phase includes mapping out these units, establishing a material flow, and integrating energy supply. By doing so, we refine efficiency and set up for economic evaluation. Remember, the design sets the stage for the cost analysis, as efficient use of resources can make or break the feasibility of the venture.

Monoclonal Antibody Production

Monoclonal antibody (MAb) production has evolved significantly, especially with advancements in cell lines like PER.C6, which offer higher yield at lower volumes. With the goal of producing 1,200 kg of MAbs annually, the focus shifts to optimizing the production scale and utilizing efficient technologies. The PER.C6 cell line facilitates a titer of 10 g/L, greatly reducing production volume and thus the size of bioreactors required.

In integrating single-use bioreactors, compliance with cleaning and sterility standards becomes less burdensome, allowing for greater flexibility and quicker setup times. The process involves culturing cells, harvesting antibodies, and then purification through successive chromatography steps, ensuring product purity.

• Affinity chromatography isolates the antibodies based on specific interactions.
• Hydrophobic interaction chromatography further removes impurities.
• An ion exchange membrane adsorber polishes the product by removing leftover charged impurities.

Understanding these steps enhances process efficiency and ensures a high-quality final product. As with any bioprocess, material and energy balances are critical to managing costs and sustainability.

Material and Energy Balances

Material and energy balances serve as the backbone of bioprocess engineering. With any given process, accurately calculating these balances is essential for resource optimization and ensuring process efficiency. In fermentation or MAb production, we need to account for every input and output. This includes raw materials like yeast, culture media, and even energy inputs like heat or chillers.

Material balances involve tracking the mass flow of inputs which translates to our product quantity – in kilograms of stevia sweeteners or MAbs. Any gain or loss must be accounted for by verifying inflows against outflows plus accumulation in the system.

Energy balances consider the thermal load required for reactions, maintaining optimal conditions, and driving separations. Efficiently using these resources minimizes operating costs and environmental impact. Solutions to exercises on this topic often involve breaking down complex processes into simpler unit operations like reactors and centrifugation, each with its own mini-balance for accuracy and simplicity.

Bioprocess Equipment Sizing

Equipment sizing is a pivotal step in bioprocess engineering, as it directly impacts process efficiency and capital investment. In designing a fermentation process or setting up for MAb production, it's crucial to determine the right size for each piece of equipment. This ensures the process can handle the desired production scale without waste or inefficiency.

For instance, a fermentation tank must accommodate not only the expected volume but also allow for headspace to prevent overflow during vigorous fermentation stages. Equipment like centrifuges, used for biomass separation, rely on process throughput, which is dictated by the fermentation production rate.

• Understand the process demands to set appropriate tank sizes.
• Utilize the product titer to gauge how much product each unit can handle.
• Adequately size ancillary systems, such as cooling towers or heaters, to efficiently support fermentation without overuse.

Optimal sizing hinges on precision and aligns directly with cost-saving measures, ensuring minimal resource wastage and investment alignment with production goals. Balancing between capital costs and operational expenses is critical to process success.