BIOCHEMICAL
`ENGINEERING
`FUNDAMENTALS
`
`James E. Bailey
`Professor of Chemical Engineering
`University of Houston
`
`David F. Ollis
`Associate Professor of Chemical Engineering
`Princeton University
`
`McGRAW-HILL BOOK COMPANY
`
`New York St. Louis San Francisco Auckland Bogota Dusseldorf
`Johannesburg London Madrid Mexico Montreal New Delhi Panama
`Paris Sao Paulo Singapore Sydney Tokyo Toronto
`
`.
`
`'
`-~
`
`Akermin, Inc.
`Exhibit 1020
`Page 1
`
`

`

`BIOCHEMICAL ENGINEERING FUNDAMENTALS
`
`Copyright © 1977 by McGraw-Hill, Inc. All rights reserved.
`Printed in the United States of America. No part of this publication
`may be reproduced, stored in a retrieval system, or transmitted, in any
`form or by any means, electronic, mechanical, photocopying, recording, or
`otherwise, without the prior written permission of the publisher.
`
`890 KPKP 83
`
`This book was set in Times New Roman. The editors were B. J. Clark,
`Barbara Tokay, and James W. Bradley; the cover was designed by
`Nicholas Krenitsky: the production supervisor was Charles Hess.
`The drawings were done by J & R Services. Inc.
`Kingsport Press, Inc .. was printer and binder.
`
`Library of Congress Cataloging in Publication Data
`
`Bailey, James Edwin, date
`Biochemical engineering fundamentals.
`(McGraw-Hill series in water resources and environmental engineering)
`(McGraw-Hill chemical engineering series)
`Includes index.
`I. Ollis, David F., joint author.
`1. Biochemical engineering.
`TP248.3.B34
`660'.63
`76-40006
`ISBN 0-07-003210-6
`
`II. Title.
`
`Akermin, Inc.
`Exhibit 1020
`Page 2
`
`

`

`176 BIOCHEMICAL ENGINEERING FUNDAMENTALS
`
`Azso
`
`As1o
`
`Chymotrypsinogen
`
`As4o
`
`0.75
`
`0.50
`
`0.25
`
`Thyroglobulin
`
`Eo
`'<j- 0
`«;tO
`II r l
`Cl
`:::..
`
`0
`
`Glucose
`
`1J
`1:::
`c
`"'
`-;
`>.
`"'
`
`1:::
`..<::
`p..
`
`0.75
`
`Cytochrome C
`
`0.50
`
`0.25
`
`54
`
`77
`
`123
`100
`Effluent volume. ml
`
`146
`
`I 71
`
`Figure 4.15 Chromatogram resulting from molecular-sieve chromatography of a solute mixture
`(packing is 6 percent cross-linked desulfated agar. Buffer is pH 7.5, 0.05 M tris-HCl). (Reprinted
`by permission from J. Porath, Chromatographic Methods in Fractionation of Enzymes, in L. B. Wingard,
`Jr. (ed.), "Enzyme Engineering," p. 154, John Wiley & Sons, New York, 1972.)
`
`needed to ensure protein stability, for example) the composition of the dialysate
`fluid in which the dialysis bag containing the sample is placed must be such that
`no finite concentration difference of these particular dialyzable components exists
`across the dialysis membrane, e.g., the use of w- 3 M phosphate buffer as dialy(cid:173)
`sate in step 7 of Example 4.1. When there are many chemicals which we wish to
`retain in the sample (such as blood), the cost of the necessary dialysate fluid may
`be very large. If a low concentration of certain dialyzable compounds is ultimately
`needed in the sample, relatively larger volumes of dialysate fluid must be used, for
`example, 6 I of phosphate buffer to dialyze 50 ml of sample (Example 4.1 ). This is
`necessary so that the concentration in the dialysate fluid will remain very small
`and there will always be a diffusive driving force. Dialysis is essentially a diffusion(cid:173)
`controlled process.
`Ultrafiltration is based upon the ability of a membrane, under a hydrostatic
`pressure head, to reject relatively high molecular weight components while pas(cid:173)
`sing both solvent and low molecular weight solutes. Thus, while dialysis generally
`removes low molecular weight molecules and ions from the original solution
`volume, ultrafiltration concentrates the original protein solution by removing
`much solvent as well as small solutes.
`With ultrafiltration, the fluxes of solvent and of solute (protein) are often
`reasonably represented by
`
`JV 1 (solvent)= LA!!p- a !in)
`JV 2 (solute)~ Cz(l - a)JV 1 + P !1C2
`
`(4.9)
`(4.10)
`
`Akermin, Inc.
`Exhibit 1020
`Page 3
`
`

`

`188 BIOCHEMICAL ENGINEERING FUNDAMENTALS
`
`Both microencapsulation methods have the potential of offering a very large
`surface area (for example, 2500 cm2 per millimeter of enzyme solution) and the
`possibility of added specificity: the membrane can be made in some cases to admit
`some substrates selectively and exclude others. In principle these methods should
`be applicable to a large variety of enzymes. However, the membrane is a
`significant mass-transfer barrier, so that the "effectiveness factor" for the enzymes
`may be quite small. Also, these techniques are not applicable when the size of the
`substrate molecule approaches that of the enzyme.
`We have already discussed (Sec. 4.2) how semipermeable-membr-ane-filtr-at-ion---(cid:173)
`devices can be used to contain enzyme while allowing interchange of smaller
`molecules with a neighboring solution. A continuous flow ultrafiltration
`concept illustrated schematically in Fig. 4.24, also contains the enzyme as in
`microencapsulation. The surface areas separating the two solutions are substanti-
`ally lower when ultrafiltration is used, and shear denaturation may occur. The
`mass-transfer rates available through membranes are small and may limit the over-
`all rate. However, almost any enzyme or combination of enzymes can be im(cid:173)
`mobilized in this way. It is advantageous when the substrate has very high
`molecular weight or is insoluble, situations where polymer-bound enzymes
`typically may have a small efficiency. Also, the semipermeable membrane will not
`permit relatively large product molecules from polymer-hydrolysis reactions to
`escape from the enzyme solution. This provides an interesting method of control-
`ling the molecular-weight distribution of the products which ultimately leave the
`reactor.
`In Sec. 4.6, we shall return to immobilized enzymes and examine some of their
`present and possible future applications. In current technology, however, enzymes
`in solution enjoy far greater use, as outlined in the next two sections.
`
`Substrate
`
`Enzyme and retentate
`
`1
`
`~--- ----
`-:-::::=..-_ ·- Enzyme-substrate-
`Reactor =---===--: ·_ :-
`product
`
`Ultrafiltration
`unit
`
`Membrane
`
`Pump
`
`Filtrate
`containing
`product
`
`Figure 4.24 Enzyme entrapment on
`a macroscale. An ultrafiltration mem(cid:173)
`brane is used to retain enzyme and
`other large molecules in the reactor.
`
`I
`
`Akermin, Inc.
`Exhibit 1020
`Page 4
`
`