LYCOBIOLOGY
`
`lfficial Journal of the Society for Glycobiology
`
`olume 19 Number 9 September 2009
`
`.glycob.oxfordjournals.org
`
`
`
`
`g.
`PROPERTY OF THE
`gm? NATIONAL
`LIBRARY OF
`MEDICINE
`
`-
`
`OXFORD UNIVERSITY PR!!!
`
`Page 1 of 21
`
`CSL EXHIBIT 1102
`
`Page 1 of 21
`
`CSL EXHIBIT 1102
`
`

`

`GLYCOBIOLOGY
`
`Editor-in-Chief
`
`Ronald L. Schnaar, The Jolnis Hopkins University.
`Ba/[jnzgfc' Mu USA
`
`Founding Editor
`Gerald W. Hart, The Johns Hopkins University
`.
`Baltimore, MD’ USA
`Associate Editor for Reviews
`
`Mark A. Lchrman, University ofTexas Southwestern Medical
`Centet; Dallas, TX, USA
`
`Associate Editors
`.
`.
`Antony BHCIC, University ofMelbourne, Melbourne, Vic", Australia
`Anne lmbcrty, CERMAV-CNRS, Grenoble, France
`Reiji Kannagi, A ic’hi Cancer Center, Nagoya, Japan
`Ulf Lindahl, Uppsala Ul'ziversity, Uppsala, Sweden
`Salvatore J . Turco, University ofKentucky, Lexington, KY, USA
`
`Editorial Board
`
`Markus Acbi, Zurich. Switzerland
`Linda G. Baum, Los Angeles, CA, USA
`C. Fred Brewer, Bronx, NY, USA
`Inka Brockhausen, Kingston, ON, Canada
`Karen J. Colley, Chicago, IL, USA
`Anthony P. Cotficld, Bristol, UK
`Paul R. Crockcr, Dundee, UK
`Richard D. Cummings, Atlanta, GA, USA
`Paul L. DeAngclis, Oklahoma City, OK, USA
`James W. Dennis, 'loronto, ON, Canada
`Kurt Driekamer, London, UK
`Alan D. Elbein, Little Rock, AR, USA
`Tamao Endo, Tokyo, Japan
`Jeffery D. Esko, La Jolla, CA, USA
`Marilynn E. Etzler, Davis, CA, USA
`Ten Feizi, Harrow, UK
`Michael A]. Ferguson, Dundee, UK
`Hudson H. Freeze, La Jolla, CA, USA
`Minoru Fukuda, La Jolla, CA, USA
`Koichi Furukawa, Nagoya, Japan
`
`John T. Gallagher, Manchester; UK
`Rita Gerardy-Schahn, Hannoven Germany
`Rudolf Geyer, Giessen, Germany
`Sen-itiroh Hakomori, Seattle, WA, USA
`Robert S. Haltiwanger, Stony Brook, NY, USA
`C‘
`a‘C.H
`"tl
`',S,~l)
`1unn ‘
`ansson, GU e mtg “a w
`Vincent C. Haseall, Cleveland, OH, USA
`Carlos B. Hirschberg, Boston, MA, USA
`Steven W. Homans, Leeds, UK
`Donald L. Jarvis, Laramie, WY, USA
`Kay-Hooi Khoo, Taipei, Taiwan
`Koji Kimata, Nagaknte, Japan
`Ken Kitajima, Nagoya, Japan
`Lena Kjellcn, Uppsala, Sweden
`‘c c"‘,"'r,D,USA
`Stcplnu L'ldm'h ”(H/””5"”!
`C
`Julie A. Leary, Davis, CA, USA
`Ludwig Lehle, Regensbzn'g, Germany
`William J. Lennarz, Stony Brook, NY, USA
`{obert J. Linhardt, 'Ii'oy, NY, USA
`Jamey D. Marth, La Jolla, CA, USA
`Malcolm J. McConville, Parkville, Australia
`Kelley W. Moremen, Athens, GA, USA
`Barbara Mulloy, Potters Bar, UK
`{afael Oriol, Villejaifl France
`Vlonica M. l’alcic, Copenhagen, Denmark
`Armando J. Parodi, Baenos Aires, Argentina
`James C. Paulson, La Jolla, CA, USA
`ichael Pierce, Athens, GA, USA
`{obert Sacksteiu, Boston, MA, USA
`Llcnrik V. Schcller, Berkeley, CA, USA
`Joel H. Shapcr, Baltimore, MD, USA
`Vathzln Sharon, Rehovot, Israel
`Pamela Stanley, New York, NY, USA
`Akemi Suzuki, Hiratsnka, Japan
`\laoyuki 'l'auiguchi, Osaka, Japan
`Maureen E. Taylor, London, UK
`Gerrit van Mcer, Utrecht, The N)t/Ierlands
`AjitVarki, LaJolla, CA, USA
`Charles J. Waechter, Lexington, KY, USA
`Katsuko Yamashita, Tokyo. Japan
`Masaki Yanagishita, Tokyo, Japan
`
`
`
`Front cover: Muscles from ma’x mice, a model for human Duchcnne Muscular Dystrophy, exhibit increased expression oi‘galectin—l dur-
`ing degeneration. Sections ofdiaphragm muscle from control (left panels) and 111ch mice (right panels) at 4 weeks of age (top panels) and
`6 weeks oi‘agc (bottom panels) are shown. Sections were stained with anti-galectin—l (green) and DAPI (nuclei, blue). Non-peripherally
`nuclcatcd myofibers, detected only in "if/X muscles, are indicated by arrowheads. Bar = 10 um. For further details, see: Ccrri DG, Rodrigues
`LC, Stowcll SR, Araujo DD, Coelho MC, Oliveira SR, Bizario JC, Cummings RD, Dias—Baruffi M, and Costa MC. 2008. Degeneration oi
`dystrophic or injured skeletal muscles induces high expression of Galcctin-l. Glycolnology. 18:842—850.
`
`Page 2 of 21
`
`Page 2 of 21
`
`

`

`eocICTy fol.
`
`" (50.“
`/
`6‘1“». ,
`‘\
`6‘
`[yea b io\°°’
`
`ANNUAL CONFERENCE
`of the
`SOCIETY FOR GLYCOBIOLOGY
`November 12 — 15, 2009
`Catamaran Resort Hotel, San Diego, CA
`
`CONFERENCE LOCATION. A11 technical sessions, posters and exhibits will be at the Catamaran Resort Hotel, 3999
`Mission Blvd, San Diego, CA 92109. The conference rate is $159 for single/double. Please refer to www.g1yc0biology.org
`for more detailed inlbrmation.
`DEADLINES
`
`Friday, July 24
`
`Friday, August 14
`Friday, August 14
`Friday, October 2
`Friday, October 9
`
`Submission of nominations for the Karl Meyer Award
`and Rosalind Kornfeld Award
`Submission of abstracts
`
`Submission of applications for Student Travel Awards
`Advance conference registration
`Reservations at the Catamaran Resort Hotel
`
`ORGANIZER
`
`Thomas N OeltIn a I] n , President and Meeting Chair
`Vanderbilt University, School of Medicine, Nashville, TN, presidentfliglycobiologrvorg or
`t_o_m.oeltInann@vandcrbilt.edu
`PRELIMINARY PROGRAM
`
`8:00 am — 5:30 pm
`
`SATELLITE MEETING: GLYCANS IN CELL COMMUNICATION
`
`Annual meeting of the Consortium for Functional Glycomics Participating Investigators
`Attendance is open to all interested scientists and isfree ofclmrge.
`Go to http://glvcomics.scrimISedu/P12009.11tml for program information
`Please notify Anna Cric (annacrieftDscripps.edu) before October 9
`SATELLITE SYMPOSIUM: ADVANCES IN GLYCOPROTEIN TECHNOLOGIES
`Orgamzed by Qun Zhou, Genzyme Corp, Cambridge, MA and Samnang Tep, BIogen ldec,
`Cambridge, MA,
`$40
`CONFERENCE OPENING
`Opening Remarks: Thomas N Oeltmann, President, Societyfor Glycobio/ogy
`SESSION I: GLYCOBIOLOGY OF THE IMMUNE SYSTEM
`
`8:30 am # 1:00 pm
`
`7:00 A 7:10 pm
`
`7:10~ 8:30 pm
`
`N
`
`:2;
`'3 ,9
`‘5 E
`5 5
`é
`
`8:30 — 9:00 pm
`9:00 — 10:00pm
`
`
`KORNFELD AWARD AND LECTURE
`
`WELCOME RECEPTION
`
`
`
`
`
`
`
`
`SESSION I]: GLYCOBIOLOGY 0F DISEASE
`8:30— 10200 am
`2
`10:30am 12:20 pm SESSION III: GLYCOBIOLOGY IN DEVELOPMENT
`Q, 3
`2:00 A 4:00 pm
`POSTERS and EXHIBITS
`72 '2
`4:00 7 5:50 pm
`SESSION 1V: GLYCOEIOLOGY 0F RECOGNITION: PROTEIN-
`L: E
`
`Z
`CARBOHYDRATE INTERACTIONS
`
`m I
`Q‘ S
`E '2
`% E
`Z
`
`8:30 — 10:00 am
`
`SESSION V: PATI-IOG EN GLYCOIIIOLOGY
`
`10:30 am — 12:20 pm SESSION VI: ASA/[B Guest Society Session: GLYCORIOLOGY AND THE
`EXTRACELLULAR MATRIX
`POSTERS and EXHIBITS
`BUSINESS MEETING
`KARL MEYER AWARD AND LECTURE
`
`BANQUET. Nominal fee. Extra tickets for guests may be ordered.
`
`2:00 — 4:00 pm
`4:0044z30 pm
`4:45 — 5:30 pm
`7:00 — 9:30 pm
`
`Page 3 of 21
`
`SESSION VII: GLYCOBIOLOGY AND THE NERVOUS SYSTEM
`8:30 # 10:00 am
`In
`10:30 am — 12:20 pm SESSION VIII: CYTOPLASMIC GLYCOSYLATION, METABOLISM, AND O—
`>2 :
`GLCNAC MODIFICATION
`8 '8
`POSTERS and EXHIBITS
`2:00 — 4:00 pm
`g E
`SESSION IX: GLYCAN RECOGNITION IN THE ER
`4:00 # 5:40 pm
`U)
`:2
`CLOSING REMARKS & RECEPTION
`5:40 — 6:00 pm
`
`Page 3 of 21
`
`

`

`DEADLINE: Friday, October 2
`ADVANCE REGISTRATION FORM
`ADVANCE REGISTER BY MAIL, FAX, 0R ONLINE. You may advance register by mailing or faxing this page (see
`below) OR on-line at wwwglycobiology.org. The deadline for advance registration is October 7 7009.
`ON SITE REGISTRATION. After October 2, please register on site. On site registration fees will be $50 more than advance
`registration. On site registration begins at 3 pm, Thursday, November 12 at the Catamaran Resort Hotel.
`
`CANCELLATIONS. Notice of cancellation must be postmarked on or before November 3, 2009. There is a $50 processing
`fee.
`
`N(line
`
`
`Company/Institul ion
`
`
`Address
`
`
`City, State, Zip/Postal Code/Country
`
`
`
`Phone
`
`Fox
`
`
`Email {receipts will be e-mailecl)
`
`SATELLITE MEETING (8 am — 5:30 pm, Thursday, November 12,_/ree ofeharge)
`Glycans in Cell Communication, Free of'ehcirge — see http://glvconiics.scrimedu/l’l200‘).litml
`
`Please notify Anna Crie (annacriefrDscrippsedu) before October 9
`
`‘
`SATELLITE SYMPOSIUM (8:30 am , 1:00 pm, Thursday, November 12, $40)
`Advances in Glycoprotein Technologies, Organized by Qun Zhou. Genzyme Corp, Cambridge, Ma
`and Samnang Tep, Biogen (doc, Cambridge, MA,
`Enter $40 for optional Satellite Syiiiposni in S
`
`CONFERENCE REGISTRATION
`
`El $400, Member ofthe Society for Glycobiology
`El $550, Non-Member
`
`'
`El $175, Student (Must be full-time in a Ph.D. program or within first two years of postdoctoral training)
`The deadline for Student Travel Awards is August 14. Go to www.gly0cobiology.org for application
`Enter registration amount S—\
`
`BANQUET TICKETS (Note! Nominal fee required) 7 pm, Saturday, November 14
`El $10, The Banquet is subsidized by the Society for Glycobiology. All registrants are enthusiastically invited to attend. A
`nominal fee is charged to assist in banquet planning. Only registered attendees are eligible for the subsidized rate.
`Enter $10 for the banquet 5
`
`EXTRA BANQUET TICKETS FOR GUESTS, 7 pm, Saturday, November 14. Registrants are eligible
`for the reduced rate (above). Guests accompanying registrants are cordially invited to attend at a cost of
`$50 per guest.
`$50 x
`number of extra tickets
`Enter extra banquet amount $
`
`ENTER TOTAL AMOUNT $
`
`On—liiie registration is available at www.glycobiology.org.
`
`El Check is enclosed for total amount. Please make check payable to Society for Glyeobiology
`El Charge total amount to my AMEX, VISA or MasterCard. Sorry, no other cards are accepted.
`
`
`Credit Card Number
`Exp (late
`
`m C
`
`Card holder ‘5 signature
`
`urd holder ’5 name
`
`Mail or Fax to:
`
`Society for Glycobiology, 2019 Galisteo Street, Building 1-1, Santa Fe, NM 87505
`Fax: (505) 989-1073 (Faxed registrations must include credit card information.)
`
`Page 4 of 21
`
`Page 4 of 21
`
`

`

`Glycobiology
`f OPEN
`3
`OXFORD *1“ y
`Volume l9 Number 9 September 2009
`
`
`Contents
`
`GLYCO—FORUM SECTION
`
`Meeting Announcements
`
`REVIEW
`
`935
`
`936
`
`950
`
`958
`
`971
`
`987
`
`995
`
`1002
`
`1010
`
`1018
`
`1034
`
`1046
`
`Optimal and consistent protein glycosylation in
`mammalian cell culture
`
`P Hosslcr, SF Khatlak, and Z] Li
`
`COMMUNICATION
`
`The specific localization of seminolipid molecular
`species on mouse testis during testicular maturation
`revealed by imaging mass spectrometry
`
`ORIGINAL ARTICLES
`
`Glycosylation profiles of cpitope-specific anti-[i-amyloid
`antibodies revealed by liquid chromatography—mass
`spectrometry
`
`Mutational and functional analysis of Large in a novel
`CHO glycosylation mutant
`Establishment of a real-time analytical method for free
`oligosaccharide transport from the ER to the eytosol
`Catabolism of flocculosin, an antimicrobial metabolite
`produced by I’seudozymaflocculosa
`Production of human B-hexosaminidase A with highly
`phosphorylated N-glycans by the overexpression of the
`()gataca minuta MNN4 gene
`'IXvo distinct (x-L-fucosidases from BUidobacterium
`bifidum are essential for the utilization of fucosylated
`milk oligosaccharides and glycoconjugates
`
`N Goto—Inoue, T Hayasaka, N Zaima, and M Setou
`
`I Perdivara, LJ Deterding, C Cozmu, KB Tomer, and
`M Przybylski
`
`JT Aguilan, S Sundaram, E Nieves, and P Stanley
`
`Y Haga, K Totani, Y Ito, and T Suzuki
`
`B Mimee, C Labbé, and RR Bélanger
`
`H Akehoshi, Y Kasahara, D Tsuji, K Itoh, H Sakuraba,
`Y Chiba, and Y Jigami
`
`H Ashida, A Miyake, M Kiyohara, J Wilda, E Yoshida,
`l-I Kumagai, T Katayama, and K Yamamoto
`
`Unusual accumulation of sulfated glycosphingolipids in
`colon cancer cells
`
`K Shida, Y Misonou, H Korekane, Y Seki, S Noura,
`M Ohue, K Honke, and Y Miyalnoto
`
`The ot-galactomannan Davanat binds galectin-l at a site
`different from the conventional galectin carbohydrate
`binding domain
`Heterodisaccharide 4-0-(N-acetyl-B-D-glucosaminyl)-
`I)-glucosamine is a specific inducer of chitinolytic
`enzyme production in Vibrios harboring chitin
`oligosaccharide deacetylase genes
`
`MC Miller, A KIyosov, and KH Mayo
`
`T Hirano, K Kadokura, T Ikegami, Y Shigeta, Y Kumaki,
`W Hakamata, T Oku, and T Nishio
`
`Page 5 of 21
`
`Page 5 of 21
`
`

`

`SUBSCRIPTIONS
`
`A subscription to Glycobiolngy comprises 12 issues. Prices include
`postage; for subscribers outside the Americas, issues are sent air freight.
`Glycobiology Advance Access contains papers that have recently been
`accepted but have not yet been included within an issue.
`
`Annual Subscription Rate (Volume 19, 12 issues, 2009)
`Institutional
`Print edition and site-wide online access: 39137 l/.£‘914/€137l
`Print edition only: $1302/f868/€1302
`Site-wide online access only: $1 l38/L‘759/€1 138
`Personal
`Print edition and individual online access: $449/..£'299/€449
`Please note: US$ rate applies to US & Canada, Euros€
`Europe, UKL’ applies to UK and Rest ofWorld.
`
`applies to
`
`There are other subscription rates available; for a complete listing,
`please visit www.glycob.0xfordjourna[sorg/subscriptions.
`
`Full pre—payrnent in the correct currency is required for all orders. Pay—
`ment should be in US dollars for orders being delivered to the USA or
`Canada; Euros for orders being delivered within Europe (excluding the
`UK); GBP sterling for orders being delivered elsewhere (i.e. not being
`delivered to USA, Canada, or Europe). All orders should be accompa—
`nied by full payment and sent to your nearest Oxford Journals office.
`Subscriptions are accepted for complete volumes only. Orders are re—
`garded as firm, and payments are not refundable. Our prices include
`Standard Air as postage outside of the UK. Claims must be notified
`within four months of despateh/order date (whichever is later). Sub~
`scriptions in the EEC may be subject to European VAT. If registered,
`please supply details to avoid unnecessary charges. For subscriptions
`that include online versions, a proportion ofthe subscription price may
`be subject to UK VAT. Subscribers in Canada, please add GST to the
`prices quoted.
`Personal rate subscriptions are only available if payment is made by
`personal cheque or credit card, delivery is to a private address, and is
`for personal use only.
`
`The current year and two previous years’ issues are available from
`Oxford University Press. Previous volumes can be obtained from
`the Periodicals Service Company, 11 Main Street, Germantown, NY
`12526, USA. E—mail: psc@periodicals.com. Tel:
`(518) 537-4700.
`Fax: (518) 537-5899.
`
`Journals Customer Service Department,
`information:
`Contact
`Oxford University Press, Great Clarendon Street, Oxford OX26DP,
`UK. E-mail: jnls.cust.serv@oxfordjournalsorg. Tel: +44 (0) 1865
`353907. Fax: +44 (0) 1865 353485.
`In the Americas, please
`contact: Journals Customer Service Department, Oxford University
`Press, 2001 Evans Road, Cary, NC 27513, USA. E-inail: jnlorders@
`oxfordjournalsorg. Tel: (800) 852-7323 (toll-free in USA/Canada)
`or (919) 677—0977. Fax: (919) 677-1714. In Japan, please contact:
`.Iotlrnals Customer Service Department, Oxford University Press, 4—
`5-10—8F Shiba, Minato-ku, Tokyo 108-8386, Japan. Email: custscrv.
`jp@oxfordjournals.org. Tel: +81 3 5444 5858. Fax: +81 3 3454 2929.
`
`(i) Cheque (payable to Oxford University
`Methods of payment.
`Press, mailed to Oxford University Press, Cashiers Office, Great
`Clarendon Street, Oxford OX2 6DP, UK) in GB J.‘ Sterling (drawn on
`a UK bank), US$19 Dollars (drawn on a US bank), or EU€ Euros. (ii)
`Bank transfer to Barclays Bank Plc, Oxford Group Office, Oxford
`(bank sort code 20—65418) (UK), overseas only Swift code BARC
`GB 22
`(GBL'
`Sterling
`to
`account
`no.
`70299332,
`IBAN
`GB89BARC20651870299332;
`USfli Dollars
`to
`account
`no.
`66014600,
`IBAN GB27BARC20651866014600; EU€
`Euros
`to
`account
`no.
`78923655,
`IBAN GB16BARC20651878923655).
`(iii) Credit card tMastercard,Visa, Switch, or American Express).
`Glycobiology is covered by the following major indexing services:
`Science Citation Index, Current Contents: Life Sciences, Reference
`Update, Mcdhne (Index Medicus), Embasc, BIOSIS.
`
`Page 6 of 21
`
`Membership in the Society for Glycobiology
`The Society for Glycobiology is a scholarly society devoted to the
`study of glycan structures and their functions.
`Individuals whosc
`professional activities are consistent with the goals of the society. as
`well as graduate students and fellows studying in the discipline, are
`invited to apply for membership online at http://www.glycobiology.
`org. Members are eligible for discounted subscription rates.
`Postal information
`
`Glycobiology (ISSN 0959-6658) is published Inonthly in Jan., Feb.
`Mar., Apr., May, Jun., Jul., Aug., Sep., Oct., Nov, and Dec. by Oxford
`University Press, 2001 Evans Road, Cary, NC 27513-2009, USA.
`Periodicals Postage Paid at Cary, NC, and additional mailing offices.
`Postmaster: send address changes to Glycobiology, Journals Customer
`Service Department, Oxford University Press, 2001 Evans Road, Cary,
`NC 275132009, USA.
`
`Oxford Journals Environmental and Ethical Policies
`Oxford Journals is committed to working with the global community
`to bring the highest quality research to the widest possible audi—
`ence. Oxford Journals will protect the environment by implement—
`ing environmentally friendly policies and practices wherever possi-
`ble. Please see http://www.oxfordjournals.org/ethicalpolicies.html for
`further information on Oxford Journals” environmental and ethical
`policies.
`
`Digital object identifiers
`For information on dois and to resolve them, please Visit www.doi.org.
`
`‘
`.
`Permissions
`For information on how to request permissions to reproduce zlrUCle
`or information from this journal, please Vistt www.oxtordjom-nulsr
`org/jills/permissions.
`
`Instructions to Authors
`Instructions to Authors can be found online at http://glycoboxford.
`journalsorg.
`
`Advertising
`Inquiries about advertising should be sent to Oxford Journals Advertis—
`ing atjnlsadvertising@oxfordjournalsorg. Tel: +44 (0) 1865 353329
`or +4401) 1865 354637.
`
`Disclaimer
`
`The author(s) of each article appearing in this Journal is (are) solcly
`responsible for the content thereof; the publication of an article shall
`not constitute or be deemed to constitute any representation by the
`Editors, the Editorial Board, the Society for Glycobiology. or Oxford
`University Press that the data presented therein are correct or suflicient
`to support the conclusions reached or that the experiment design or
`methodology is adequate.
`
`Copyright © 2009 Oxford University Press
`
`All rights reserved: 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 prior written permission of the publisher or a license permit-
`ting restricted copying issued in the UK by the Copyright Licensing
`Agency Ltd, 90 Tott‘enham Court Road, London W1 P 9HE, or in the
`USA by the Copyright Clearance Center, 222 Rosewood Drive, Dati-
`vers, MA 01923. For those in the USA or Canada not registered with
`CCC, articles can be obtained by fax in 48 hours by calling: WISE for
`Medicine’l‘M 1—800-667-WISE.
`
`is a department of the University of
`Oxford University Press
`Oxford. It furthers the University’s objective ofexccllcnce in research,
`scholarship, and education by publishing worldwide.
`
`Page 6 of 21
`
`

`

`Glycolfiology vol. 19 no, 9 pl 935, 2009
`doi:10‘1093/glycob/cwp108
`
`Glyeo—Forum section
`
`
`
`Meeting Announcements
`
`6th International Conference on
`
`Proteoglycans
`
`September 13—17, 2009, Aix-les-Bains, France
`
`Conference co-m‘ganizers:
`H. Lortat-Jacob (Grenoble, France)
`J. Van den Born (Groningen, The Netherlands)
`Contact: contact.pg2009@ibs.fr
`
`For details please visit: http://pg2009-france.ibs.fr/.
`
`2009 Annual Meeting of the Society for
`Glycobiology
`
`November 12—15, 2009, Catamaran Hotel, San Diego,
`California
`
`Program Chair: Tom Oeltmann, Vanderbilt University
`For details please visit http://www.glycobiology.org
`
`20‘“ International Symposium on
`Glycoconjugates
`Caribe Hilton Hotel, San Juan, Puerto Rico
`November 29—December 4, 2009
`
`For further details, please visit http://www.glyco20.org.
`
`Dr. Dipak K. Banerjee
`Department of Biochemistry
`School of Medicine, University of Puerto Rico
`Medical Sciences Campus
`GPO Box 365067, San Juan, PR 00936—5067
`Telephone: +1-787—758-2525 ext. 1624 or +1-787-758-7090
`Fax: +1-787-274-8724
`
`Email: dbanerjee@rcm.upr.edu (Scientific Secretariat) or
`ccastaneda@lecmanagcmentcom (Organizational Secretariat)
`
`Published by Oxford University Press 2009.
`
`Page 7 of 21
`
`935
`
`Page 7 of 21
`
`

`

`Glycobiology vol. 19 no. 9 pp. 936-949, 2009
`doi:10.1093/g1ycob/ewp079
`Advance Access publication on June 3, 2009
`
`REVIEW
`
`Optimal and consistent protein glycosylation in mammalian cell culture
`
`Patrick Hossler3'4’2, Sar 'at F Khattak“, and
`Zheng Jian Lil’4
`
`4Process Sciences Upstream, Technical Operations, Bristol-Myers Squibb
`Company, East Syracuse, NY 13057, USA
`
`Received unA/Jri/ 6, 2009; revised on June I, 2009; accepted on Jame I, 200‘)
`
`In the biopharmaeeutical industry, mammalian cell culture
`systems, especially Chinese hamster ovary (CHO) cells, are
`predominantly used for the production oftherapeutic glyco-
`proteins. Glycosylation is a critical protein quality attribute
`that can modulate the efficacy of a commercial therapeu—
`tic glycoprotein. Obtaining a consistent glycoform profile in
`production is desired due to regulatory concerns because a
`molecule can be defined by its carbohydrate structures. An
`optimal profile may involve a spectrum of product glycans
`that confers a desired therapeutic efficacy, or a homogeneous
`glycoform profile that can be systemically screened for. Stud-
`ies have shown some degree of protein glycosylation control
`in mammalian cell culture, through cellular, media, and pro-
`cess effects. Studies upon our own bioprocesses to produce
`fusion proteins and monoclonal antibodies have shown an
`intricate relationship between these variables and the result-
`ing protein quality. Glycosylation optimization will improve
`therapeutic efficacy and is an ongoing goal for researchers
`in academia and industry alike. This review will focus on
`the advancements made in glyeosylation control in a manu-
`facturing process, as well as the next steps in understanding
`and controlling protein glycosylation.
`
`Keywords: bioproccssing/mammalian cell culture/protein
`glycosylation/protein therapeutics/protein quality
`
`Introduction
`
`Protein glycosylation is of paramount importance to the efficacy
`and manufacturing of therapeutic glycoproteins. Mammalian
`cell expression systems are the preferred method for the com-
`mercial production of these glycoproteins because their innate
`protein processing machinery, including that of protein glyco—
`sylation, closely resembles that in human.
`Glycosylation of proteins takes on the form of oligosaccha—
`rides attached to either the side chain of asparagine (N-linked)
`or serine/threonine (0-1inked) with the former being the most
`
`
`ITo whom correspomlencc should be addressed: Tel: + 1-315-432-2131 ; e-mail:
`zlicngjian.1i@bms.com
`2Present address: Abbott Biorcsearch Center, Abbot Laboratories, Worcester.
`MA 01605, USA,
`3These authors contributed equally to this work.
`
`prominent (Warren 1993; Helenius and Aebi 2001; Sinclair and
`Elliott 2005). Glycans have a very prominent role toward af-
`fecting therapeutic efficacy and determining the in vivo half-life
`(Elliott et al. 2003). It is due to both of these features that the
`glycoform profile of a therapeutic glycoprotein must be exten-
`sively characterized in order to meet regulatory agency demands
`(FDA 1996).
`In this review, we summarize the salient features of protein
`glycosylation control from a bioprocess perspective. Through
`our own process development and characterization efforts with
`fusion proteins such as OrcnciaTM and Belatacept and motio-
`clonal antibody fpilimumab, we have gained an appreciation
`over the past, present, and future state of the control of this
`very important metabolic pathway. Table I lists glycosylated
`therapeutics currently on the market, which is the impetus
`for understanding their metabolic control. Although CHO cells
`are the most prevalent for producing glycoprotein therapeutics,
`other cell lines such as human embryonic kidney cells (HEK)
`(Duroeher ct al. 2007), baby hamster kidney (BHK), mouse
`myeloma (N50), and human retinal cells (PERC.6)
`(Jones
`et al. 2003; Wurm 2004; Petricciani and Sheets 2008) are being
`developed as high producing cell lines.
`There have been numerous reports on the effects of cell type,
`culture process, and culture media toward the resulting glyco-
`form profile. This review will summarize these efforts, as well
`as new attempts toward achieving uniform, consistent, and/or
`optimal glycosylation. These efforts share a common need for
`a more fundamental understanding of the metabolic pathway,
`which is the next logical progression from the current paradigm
`of glycosylation maintenance. The ultimate goal would be to
`take the lessons learned front previous studies, combined with
`new technologies, to transform a well-characterized cell line to-
`ward one that can control the glycosylation profile. In order to
`truly incorporate process quality by design (QbD) into the final
`glyeoprotein product (Rathore and Winkle 2009), more funda—
`mental studies on the dependence of glycosylation on process
`conditions and cellular physiology will be required to gain phys-
`iological insight. The information gathered from such research
`together with clinical studies will continue to enhance our capa-
`bility toward generating highly efficacious glycoprotein drugs.
`
`Protein glycosylation and product quality
`
`Metabolic pathway
`The addition of oligosaccharides onto a glycoprotein is a com-
`plex metabolic pathway, characterized by the en bloc transfer
`of polysaccharide chains, as well as the step-wise addition and
`removal of individual monosaccharides. The number of path-
`ways traversed is dependent on reaction site accessibility, the
`
`© The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please c-mail: journalspermissions@oxterdjournals.org
`
`936
`
`Page 8 of 21
`
`Page 8 of 21
`
`

`

`
`
`Table I. Glycosylated therapeutics approved by the FDA and/or EMEA
`
`Product
`Class
`Mode of action
`Indication
`Cell litre
`Company
`
`
`Optimal protein glyeosylation in mammalian cell culture
`
`Regulates red blood cell production
`
`Anemia
`
`(.‘ryoporin-associatcd
`periodic syndromes
`Colorectal cancer
`
`CHO
`
`CHO
`
`CHO
`
`Arngen
`
`Regeneron
`
`Genentech
`
`Crohn’s disease &
`Remieade
`Centocor/Johnson
`Rheumatoid arthritis
`(Intliximab)
`& Johnson
`Non-Hodgkins
`Binds to the cluster of dilfcrcntiation 20 (CD20)
`Rituxan
`rMab
`CHO
`Gencntcch (1 DEC)
`lymphoma
`which is expressed on B-eclls. Fc portion
`(Ritnximab)
`mediates ADCC and CDC
`
`Arancsp
`(Darbepoetin
`alfa)
`Arealyst
`(Rilonacept)
`Avastin
`(Bevaeizumab)
`Avoncx
`(Interferon
`fi—la)
`Cerczynie
`(lmiglucerasc)
`
`Erythropoiesis
`stimulating
`protein
`lL-l Trap
`
`rMab
`
`interferon
`
`Enzyme
`
`Elaprase
`(lduronate
`sulfatase)
`
`Enbrel
`(Etancreept)
`
`Epogen (lipoetin
`alfa)
`
`Erbilux
`(Cctuximab)
`
`lIcrccptin
`('l'rastuzumal))
`Wellferon
`(Interferon u)
`
`Mirecra (Mcthoxy
`polyethylene
`glycol-cpoetin
`beta)
`Myozymc
`(Alglucosidasc
`alfa)
`Naglazyme
`(Galsult‘asc)
`
`NeoRccorrnon
`(Epoetin bcta)
`
`Orencia
`(Abatacept)
`
`l’rocr'it/[Eprex
`(Epoctin alto)
`
`Enzyme
`
`Fusion protein
`
`Erythropoicsis
`stimulating
`protein
`
`rMab
`
`rMab
`
`Cytokine
`
`Erythropoicsis
`stimulating
`protein
`
`Enzyme
`
`Enzyme
`
`lirythropoicsis
`stimulating
`protein
`
`Fusion Protein
`
`Erythropoicsis
`stimulating
`protein
`
`Rebif (Interferon
`{9- I a)
`
`Cytokinc
`
`rMab
`
`Binds lL-li‘i to prevent the interaction to cell surface
`reccptors
`Binds to the vascular endothelial growth factor
`(VEGF) to inhibit angiogencsis
`Binds to type I interferon receptors to activate two
`.lak tyrosine kinases
`
`Engineered to have mannose-tcrrninated
`oligosaccharide chains that are recognized by
`etttlocytic carbohydrate receptors on
`macrophages. Catalyzcs hydrolysis of glycolipid
`glucoeerebrosidc to glucose and ccramidc in
`those macrophages which accumulate lipids in
`Gaucher disease
`llydrolyzes the 2-sull‘atc esters of terminal iduronate
`sulfate residues from the glycosaminoglycans
`dcr'matan sulfate and heparan sulfate in the
`lysosomes of various cell types
`Mimics inhibitory effects of naturally occurring
`soluble TNF receptors to rcdtrec intlammatory
`l'ChpUllSU
`Recombinant human erythropoictin interacts with
`eryt iropoietin (EPO) receptors to stimulate
`production of red blood cells froiu bone marrow
`stem cells
`
`Binds o the extracellular domain of the epidermal
`growth factor (EGFR) preventing activation of
`EGFR to impair cell growth and proliferation
`Binds o llER2+ tumor cells, blocks downstream
`HERZ signaling to inhibit proliferation of cells
`Binds 0 type I interferon receptors (IFNARI and
`lFNAch) which, upon dimcrization, activate two
`Jak Janus kinase) tyrosine kinascs (.lakl and
`'l‘yk2). Uprcgulates expression of MHC 1 proteins
`Recombinant human erythropoictin interacts with
`cryt iropoietin (EPO) receptors to stimulate
`prot action of red blood cells from bone marrow
`stem cells
`Recombinant acid (ngllCOhltillSC (GAA) to replace
`GAA deficiencies
`
`
`
`Recombinant form of polymorphic human enzyme
`Niacctylgalactosamine 4—sull‘atasc which
`catabolizes glycosarninoglycans (GAG)
`Recombinant human erythropoictin interacts with
`erythropoictin (EI’O) receptors to stimulate
`production of red blood cells from bone marrow
`stem cells
`C'l‘LA-i-lg acts as a selective modulator of the
`costirntilatory signal required for full 'l'—eell
`activation
`Recombinant human erythropoictin interacts with
`erythropoictin (El’O) receptors to stimulate
`production of red blood cells from bone marrow
`stem cells
`lrnmunomodulation through the induction of cell
`membrane components of the major
`histocorupatibility complex
`Binds to 'I‘NF and inhibits TNF action
`
`Multiple sclerosis (MS)
`
`Cl l()
`
`Biogcn Idcc
`
`Gaucher Disease
`
`CllO
`
`Genzyme
`
`l-Iuntcr syndrome
`
`[if-1080
`
`Shire
`
`Rheumatoid arthritis
`
`CHO
`
`Amgcu
`
`Anemia
`
`CIIO
`
`Amgen, Kirin
`
`Colorcclal cancer
`
`Sl’2/0
`
`lrnclone/BMS
`
`Breast cancer
`
`CHO
`
`Chronic hepatitis C
`
`Human
`lympho-
`blastoid
`
`Anemia
`
`Pompe disease
`
`Mucopolysaccharidosis
`VI (MI‘S Vt)
`
`Anemia
`
`CHO
`
`CHO
`
`CHO
`
`CHO
`
`Rheumatoid arthritis
`
`CHO
`
`Anemia
`
`CHO
`
`Multiple sclerosis (MS)
`
`CHO
`
`GCHCHYCCII
`
`Wellcornc
`
`Hoffmann-La
`Roche
`
`Genzyme
`
`Biomarin
`
`Roche
`
`Bristol—Myers
`Squibb
`
`10"“5011 3L
`JOhnSOlL
`Schering-
`PlOllgh
`MCI'Ck Scrono
`
`51’2/0
`
`Page 9 of 21
`
`(Continued)
`
`937
`
`Page 9 of 21
`
`

`

`l’ Hossler et al.
`
`Table 1. (Continued)
`
`Product
`Class
`Mode of action
`
`
` Indication Cell line Company
`
`
`
`Soiiris
`(Eculizumab)
`
`rMab
`
`Synagis
`(Palivizumab)
`TNKase
`(Teneeteplase)
`(IPA)
`
`Tysabri
`(Natalizumab)
`
`Vectibix
`(Paniturnumab)
`
`Zerrapax
`(Daclizumab)
`
`rMab
`
`Enzyme
`
`rMab
`
`rMab
`
`rMab
`
`Binds to the complement protein C5, inhibiting
`terminal complement mediated intravascular
`hcmolysis
`Targets an epitope iii the A antigenic site of the F
`protein of respiratory syncytial virus (RSV)
`Recombinant fibrin—specific plasminogen activator
`
`Paroxysmal nocturnal
`hemogiobinuria
`
`Respiratory syncytial
`virus
`Acute myocardial
`infarction
`
`C110
`
`NSO
`
`CHO
`
`Alcxion
`
`Mcdlmmune
`
`(ienctcch
`
`Binds to 014 integrin of adhesion molecule VLA-4
`and sterieally inhibits binding of VLA-4 to
`VCAM~1
`
`Multiple sclerosis (MS)
`
`C110
`
`Biogen Idec
`
`Binds to EGFR
`
`Binds to the or subunit (p55 or, CD25, or Tac subunit)
`of human lL—2 receptor expressed on the surface
`of activated lymphocytes
`
`Colorectal carcinoma
`
`Acute organ rejection
`
`CHO
`
`Sl’2/(l
`
`Aingen
`
`I'lot'fmann-La
`Roche
`
`localiza—
`enzymatic substrate specificities, as well as spatial
`tion of the various enzymes and nucleotide—sugar substrates that
`are necessary for the reactions to proceed in a particular order
`(Krambeck and Betenbaugh 2005; Butler 2006).
`Whereas N—linked glycosylation initiates primarily in the ER
`of mammalian cells, 0-1inked glycosylation has been shown
`to initiate in either the ER or Golgi apparatus. Typical N— and
`O-glycan structures with their symbolic monosaceharide repre-
`sentation and nomenclature are shown in Figure i.
`There exists the potential for a diverse array of product gly-
`cans on each glycoprotein molecule as demonstrated in the
`KEGG GLYCAN database (Hashimoto et at. 2006). In most
`industrial bioprocesscs, this large repertoire of glycans is typi—
`cally not seen on recombinant glycoproteins expressed by mam-
`malian cells such as CH0 and N80, suggesting that there may
`be pathway constraints. Control of this metabolic pathway can
`be manifested through a variety of different conditions, includ—
`ing the cell lines, bioprocess, and cell culture media used for
`protein production.
`
`Manosaccharide and ()li'gosacc/tclricle roles in protein
`therapeutic ejj’icucy
`
`N— aird O-linked glycans have been shown to have a large effect
`on the immunogenicity, efficacy, solubility, and half-life ofcom-
`mercial biologics (Lis and Sharon 1993; Van den Steen et al.
`1998; Lowe and Marth 2003). Individual monosaceharides on
`N— and O-glycan structures can help determine a glycoprotein’s
`therapeutic efficacy by their immunological and pharmacoki-
`netic impact (Sethuraman and Stadheim 2006).
`
`N—linkezl Glycosy/utiort. Figure 2 is a schematic of N—linked
`glycosylation fora protein therapeutic in a CHO cell culture.
`The terminal N—acetylneuraminic acid (NANA) content in a
`glycoprotein has been shown in many cases to be intricately
`linked to circulatory half-life, and thus is a typic