Homework+8 - Homework 8: Due Friday, Dec. 3rd...

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Unformatted text preview: Homework 8: Due Friday, Dec. 3rd Chemical Engineering 170A: Biochemical Engineering Question 1. Evaluation of Filtration Constants A fermentation broth was filtered at a constant pressure ΔP = 338 kPa. Data for the volume of filtrate collected is shown below: t (sec) V (L) 0 0 4.4 0.498 9.5 1 16.3 1.501 24.6 2 34.7 2.498 46.1 3.002 59 3.506 73.6 4.004 89.4 4.502 107.3 5.009 Calculate the constants α and Rm from the data given. A = 0.0439 m2 ρ0 = 23.47 g cells/L μ= 8.937x10 ­4 kg/(m sec) Hint: A plot of 1/(dV/dt) vs V may be useful. Question 2. Chromatography Consider the chromatographic separation of two proteins (X and Y) on an ion exchange column (length = 1m, ε= 0.25). A pulse injection of 10mL containing 10 mg/ml of X and 5 mg ml ­1 of Y enters the column. The interstitial velocity is 20 cm min ­1. The density of the particles is ρP = 2.0 g ml ­1. The adsorption of protein X follows a linear isotherm q* = ACx q* is mass solute per mass particle, Cx is mass solute per liquid volume, and A is 0.85. The units for A are mL liquid per g particle. The adsorption of protein Y follows a power isotherm q*= B (Cy)n q* is mass solute per mass particle, Cy is mass solute per liquid volume, B is 0.60, and n=2. The units for B are mL liquid mL solute per g solute per g particle. Using the Fundamental Equation of Chromatography, calculate the average retention time of proteins X and Y. Question 3. Protein Purification Studies have shown that your desired product protein (Protein 1) is prone to aspartic acid hydrolysis, and the resulting isoaspartyl peptide is not biologically active. To minimize the rate of hydrolysis, the protein must be maintained at pH 8.5 throughout the purification process. After preliminary purification steps, two contaminating proteins remain (Proteins 2 and 3). You plan to use ion exchange chromatography to remove these remaining contaminants. Net Charge on Protein 5 4 3 2 1 0  ­1 0  ­2  ­3  ­4  ­5 Protein 1 Protein 2 Protein 3 pH 2 4 6 8 10 12 14 Electrophoretic Titration Curve a. Based on this electrophoretogram, do you recommend anion or cation exchange for the separation? b. Based on the electrophoretogram, which protein elutes in the first peak of this gradient elution ion exchange chromatogram. c. Based on the gradient elution data, what salt concentration would you use to load a manufacturing column (to be operated in step mode) if you wish to capture the contaminants while allowing the product protein to flow through (without binding)? Salt (mM) 500 400 300 200 100 0 0 5 10 15 Column Volumes 20 Gradient Elution pH 8.5 Question 4. Your team has been asked to develop a purification process for a new therapeutic protein to be used in clinical trials. After cell removal and cation exchange chromatography, the product (“P”) is approximately 70% pure, contaminated by two protein species (“A” and “B”) not related to the product. The protein “B” can be very efficiently removed by strongly binding in anion exchange chromatography, but the proteins “A” and “P” co ­elute (are not separated from each other) in anion exchange. The figure below shows the result of your work optimizing a laboratory hydrophobic interaction chromatography (HIC) step using Phenyl Sepharose resin and a gradient of (NH4)2SO4 in 100 mM NaCl, 20 mM NaPO4 at pH 7.0. This HIC step separates the three proteins (“A” is in the leading peak, “P” in the middle peak, and “B” in the final peak). Unfortunately, the HIC step does not achieve baseline separation, so that the “P” peak is contaminated by traces of “B” (beginning at 67 minutes), and product “P” is found at significant levels in the “B” peak (out to 80 minutes). You plan to use a sequence of the three chromatography steps to manufacture the product. Optimized HIC Laboratory Gradient Elution 1200 1000 800 600 400 200 0 0 20 40 60 80 100 120 Time (min) 1. List the order of the chromatography steps that you recommend for manufacturing. 2. Fill in the blanks in this procedure to indicate what (NH4)2SO4 concentration you recommend for: a) equilibration, b) washing, c) elution, in the HIC manufacturing step (using step elution). HIC Production Step Elution Procedure Regenerate/Equilibrate: ______ mM (NH4)2SO4, 100 mM NaCl, 20 mM NaPO4, pH 7.0. 10 Column Volumes. Load Product: (NH4)2SO4 & pH pre ­adjusted to match column. (NH4)2SO4 (mM) Wash: ______ mM (NH4)2SO4, 100 mM NaCl, 20 mM NaPO4, pH 7.0. 5 Column Volumes. Elute Product: ______ mM (NH4)2SO4, 100 mM NaCl, 20 mM NaPO4, pH 7.0. Collect UV Positive Peak Clean: Water ­for ­Injection (Distilled Water). 5 Column Volumes. Sanitize: 1 M NaOH. 3 Column Volumes Hold for 30 minutes Rinse with 3 Column Volumes Water ­for ­Injection Question 5. Your company’s manufacturing facility requires a process to clarify the broth from a bioreactor to produce 10,000 L of cell ­free harvest for antibody purification. The plant has an existing filtration system with a positive displacement pump of 200 L/min capacity used to circulate the culture fluid. The system accommodates flat plate cartridges connected in parallel. Each cartridge has 20 channels (14 cm wide and with 0.42 mm between plates) giving a total membrane surface area of 1.7 M2 for each cartridge. Bench scale tests show that the culture broth is effectively clarified with minimal loss of antibody by using 0.65 um pore size polyvinylidene fluoride membranes, at a wall shear rate of 2,000 sec ­1 and a flux of 50 L/M2/hr. a) For a wall shear rate of approximately 2,000 sec ­1, how many filter cartridges can the existing pump support? b) If the filtration must be completed in 6 hours (to fit reliably in one 8 ­hour work shift), is a new pump (of larger capacity) needed? Question 6. Constant Temperature Continuous Sterilization Blanch and Clark Ch 5 Problem 11 Question 7. Forces in a Centrifuge A centrifuge bowl is spinning at a constant 2000 rpm. What radius bowl is needed for the following? (a) A force of 455 g's (b) A force four times that in part (a) ...
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This note was uploaded on 01/28/2011 for the course CHE 170A taught by Professor Blanche during the Spring '10 term at Berkeley.

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