Lecture Notes 3

Lecture Notes 3 - Concept Question(s) for Cell lysis Click...

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Click to edit Master subtitle style 8/9/11 Concept Question(s) for Cell lysis
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8/9/11 Valve-type high pressure homogenizer Bench scale unit from Niro-Soavi Pressure adjusting handwheel
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8/9/11 CONCEPT QUESTION 1 For a high-pressure homogenizer, ln(1-R) = -k N Pα where R is fraction of cells lysed, N is number of passes, P is pressure, and α and k are a constants for a given stream and device. If 80 % of the cells get lysed in the first pass, how many cells survive 3 passes? A (red): 5 % B (yellow): 2 % C (green): 1 %
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8/9/11 Answer to Q1 Ans C (green): 1 % Note the log of the surviving fraction is proportional to number of passes. So same percentage of entering stream survive each pass (20 %). 1 pass = 20 % alive; 2 passes = 0.2 * 0 .2 = 4 % alive; 3 passes = 0.2 * 0.2 * 0.2 = 1 %. Model is “black and white” w r t
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8/9/11 Graphical view Ln (1-R) or ln (S) N 0 (S = 100 %) Slope = - k P α = - 1.6 -4.8 (S = 1 %) 0 3 Ln (0.2) = -1.6 (S = 20 %) 1
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8/9/11 Osmotic pressure – it’s real Salty water Pure water At equilibrium, chemical potential of water on both sides is the same. Higher pressure needed on salty side to compensate for its lower concentration so as to give the right chemical potential. OP is a colligative property – it depends on the total conc of all solutes and is not very dependent on the nature of the solutes. Tricky point – The van’t Hoff law expresses osmotic pressure in terms of TOTAL conc of all solutes. A 100 mM aq NaCl soln has a total solute conc of 200 mM. Membrane permeable only to water
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8/9/11 What the textbook missed… Lipopolysaccharides (Gram negative bacteria) are pyrogenic! LPS are a k a endotoxins, Injectible protein drugs typically have < 1 EU = 1 ng LPS in a dose. effectively removed by “Q” ion exchange resin in column format, presumably via phosphate groups of LPS.
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8/9/11 Penquin Question (Q2) – inline treatment of It is not uncommon for the lysate leaving a homogenizer to be chilled and/or the pH adjusted in-line . Offer a rationale for why processes would add these measures.
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8/9/11 Answer to Q2 Lysis liberates peptidases and other unpleasant enzymes that can attack the product in the lysate. It can be a bad neighborhood for the product. Chilling slows down the enzyme kinetics, like for any other chemical reaction Enzyme rxs are pH sensitive so adjusting pH can also help Sometimes metal chelators are also
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8/9/11 Penquin Question 3 Your bioprocess makes an injectible antibiotic. Imagine a scenario where there are two seemingly equal technologies for performing lysis of the host cell: (1) high pressure homogenizer, or (2) adding an organic solvent, toluene.
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Lecture Notes 3 - Concept Question(s) for Cell lysis Click...

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