91Ó°ÊÓ

Mammalian cells can be cultured for a variety of purposes, including synthesis of vaccines. They must be maintained in growth media containing all of the components required for proper cellular function to ensure their survival and propagation. Traditionally, growth media were prepared by blending a powder, such as Dulbecco's Modified Eagle Medium (DMEM) with sterile deionized water. DMEM contains glucose, buffering agents, proteins, and amino acids. Using a sterile (i.e., bacterial-, fungal-,and yeast-free) growth medium ensures proper cell growth, but sometimes the water (or powder) can become contaminated, requiring the addition of antibiotics to eliminate undesired contaminants. The culture medium is supplemented with fetal bovine serum (FBS) that contains additional growth factors required by the cells. Suppose an aqueous stream (SG = 0.90) contaminated with bacteria is split, with 75\% being fed to a mixing unit to dissolve a powdered mixture of DMEM contaminated with the same bacteria found in the water. The ratio of impure feed water to powder entering the mixer is 4.4:1. The stream leaving the mixer (containing DMEM, water, and bacteria) is combined with the remaining 25\% of the aqueous stream and fed to a filtration unit to remove all of the bacteria that have contaminated the system, a total of \(20.0 \mathrm{kg}\). Once the bacteria have been removed, the sterile medium is combined with FBS and the antibiotic cocktail PSG (Penicillin-Streptomycin-L-Glutamine) in a shaking unit to generate 5000 L of growth medium (SG = 1.2). The final composition of the growth medium is 66.0 wt\% H_O, 11.0\% FBS, 8.0\% PSG, and the balance DMEM. (a) Draw and label the process flowchart. (b) Do a degree-of-freedom analysis around each piece of equipment (mixer, filter, and shaker), the splitter, the mixing point, and the overall system. Based on the analysis, identify which system or piece of equipment should be the starting point for further calculations. (c) Calculate all of the unknown process variables. (d) Determine a value for (i) the mass ratio of sterile growth medium product to feed water and (ii) the mass ratio of bacteria in the water to bacteria in the powder. (e) Suggest two reasons why the bacteria should be removed from the system.

Step by step solution

01

Drawing and labeling the process flowchart

Start by choosing symbols to represent the components involved in the process, such as a line for connecting elements and circles for units of operation. As described, the flow chart should contain a stream split into two, with one part going to a mixing unit and the other part combined post-mixing. The mixed stream, consisting of DMEM, water, and bacteria, is fed into a filtration unit, after which it is mixed with FBS and PSG in a shaking unit. The final product is a growth medium.

02

Degree-of-feedom analysis

This step aims to determine the unknown variables for each piece of equipment. For each system, the degree of freedom (DOF) is calculated by the formula: DOF = n – e + 1, where n is the number of unknowns or variables and e is the number of independent equations. According to this, you'll have a specific DOF for the mixer, filter, shaker, splitter, the mixing point, and the overall system.

03

Calculating process variables

This step involves using mathematical techniques to solve for every unknown variable. Given the information about various inputs and outputs, together with known principles or equations (like mass balance), you should try to derive every unknown variable involved in the system.

04

Determining the mass ratio

This involves determining (i) the mass ratio of sterile growth medium product to feed water and (ii) the mass ratio of bacteria in the water to bacteria in the powder. Based on mass balance, the mass ratios can be calculated.

05

Reasons for bacteria removal

This is a reasoning step, and it should include two reasons for bacteria removal. An example of this could be to ensure the medium remains sterile and conducive to the growth of mammalian cells, and to prevent contamination that could alter the cell's function or damage the cells.

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Key Concepts

These are the key concepts you need to understand to accurately answer the question.

Mammalian Cell Culture

Mammalian cell culture involves the growth and maintenance of mammalian cells in a controlled environment. These cells are used for a variety of scientific and medical purposes, including vaccine production and research. To successfully grow mammalian cells, several essential conditions need to be met:

- A nurturing environment is essential, which mimics the natural conditions of the cells. - Nutrient-rich growth media is required, often supplemented with fetal bovine serum (FBS) which provides growth factors. - Sterility is crucial to prevent contamination that could damage the cells or alter their function.

Mammalian cell culture is a delicate process, requiring a balance of nutrient supply and contamination control to ensure that cells grow healthily and according to research or production requirements.

Degree-of-Freedom Analysis

Degree-of-freedom (DOF) analysis is a fundamental concept in chemical engineering, used to analyze systems of equations in regards to process variables. Typically expressed by the equation:\[ \text{DOF} = n - e + 1 \]where \( n \) is the number of unknown process variables and \( e \) is the number of independent equations available, this equation allows engineers to determine whether a system is solvable. If the DOF is zero, the process is deterministic and solvable. A positive DOF indicates a need for additional equations or data, while a negative DOF means the system is over-specified.

In the context of mammalian cell culture, DOF analysis helps identify which parts of the overall process—such as mixers, filters, or shakers—can be further explored to determine unknown process variables. This is important to ensure efficient design and operation of the chemical processes involved.

Sterile Growth Media

Sterile growth media is vital for supporting the healthy development of mammalian cells. This media provides essential nutrients and serves as a controlled environment free from unwanted microorganisms. The growth media consists of several key components:

- Water, which acts as a solvent and medium for reactions. - DMEM, a common powder containing sugars, amino acids, and other nutrients. - Fetal bovine serum (FBS) and antibiotic cocktails such as PSG to support growth and prevent contamination.

Maintaining sterility is paramount, as even slight bacterial, fungal, or yeast contamination can compromise cell growth. This is resolved through processes like filtration which remove unwanted microorganisms to ensure the medium remains conducive to cell culture.

Process Flowchart

A process flowchart is an essential tool in chemical process design. It visually represents the steps and flow of materials through a system. In the context of mammalian cell culture, a flowchart may include:

- Splitting of aqueous streams and their mixing with powdered substances in a mixer. - The movement of combined streams through a filtration unit to remove contaminants. - The final mixing with additional components like FBS and antibiotics to form a sterile culture medium.

The flowchart helps identify the sequence of processing units and materials involved, aiding in the understanding of how each part of the process contributes to the final product. It is crucial for engineers to draw precise, labeled flowcharts to efficiently communicate the setup and interactions within a chemical process.