AF Labrijn

Alexander G Tulloch

Amareth Lim

Ambrose Clarke

B Mullan

Brian Mullan

Bruce Ricart

Bryan Dravis

Don Olson

J Wypych

M Trexler-Schmidt

M van der Neut Kolfschoten

Pete Lambooy

Susan Janes

Tomas O’Riordan

Y-H Kao

YD Liu

English

PubMed

Springer - Publisher Connector

MEETING ABSTRACT Open Access
Disulphide bond reduction of a therapeutic
monoclonal antibody during cell culture
manufacturing operations
Brian Mullan1*, Bryan Dravis2, Amareth Lim3, Ambrose Clarke4, Susan Janes3, Pete Lambooy2, Don Olson2,
Tomas O’Riordan1, Bruce Ricart3, Alexander G Tulloch2
From 22nd European Society for Animal Cell Technology (ESACT) Meeting on Cell Based Technologies
Vienna, Austria. 15-18 May 2011
Background
Disulphide bonding is critical to maintaining immuno-
globulin (IgG) tertiary and quaternary structure for ther-
apeutic monoclonal antibodies (MAb). Both inter- and
intra-chain disulphide bonds are formed intracellularly
in the expression host prior to secretion and purification
during MAb production processes. Disulphide bond
shuffling has previously been reported for IgG2[1,2] and
disulphide-mediated arm-exchange for IgG4[3,4], reflect-
ing innate behaviour of these IgG classes. However, aty-
pical and significant reduction of disulphide bonds has
been recently observed in IgG1[5,6] that present signifi-
cant issues for manufacturing of therapeutic MAbs.
During manufacturing of preliminary lots of a recently
transferred MAb manufacturing process (IgG1), gross
disulphide bond reduction following affinity capture
chromatography of clarified production bioreactor mate-
rial was observed. Investigations leading to the identifi-
cation of the nature of this reduction process, and
process steps to mitigate against its future occurrence,
are described here. The MAb was co-developed with
MacroGenics, Rockville, MD.
Methods
Production Bioreactor material for downstream proces-
sing was supplied from a 16 day, fed-batch, GS-CHO
culture [7]. The Production Bioreactor was a single-use
(Wave System) with a 100L (full scale) or 10L (lab
model) working volume. Clarified harvest intermediate
(CHI) hold studies were performed in either 560L Lev-
Mix units (full scale) or 5L Braun benchtop bioreactors
(lab model). Purification to produce Affinity Capture
chromatography eluted mainstream was performed
using a 20cm x 20cm MAbselect Protein A resin (GE
Healthcare) column, and an AKTA Process skid (GE
Healthcare).
LC-MS analysis was performed on a Polymer Labora-
tories PLRP-S HPLC column and analyzed using an Agi-
lent 1100 HPLC system coupled to an Applied
Biosystems QSTAR XL mass spectrometer, following
sample preparation. CE-SDS analysis was performed
using a Beckman Coulter PA800 capillary electrophor-
esis instrument fitted with bare-fused silica capillary and
UV detection at 220 nm, following sample preparation.
Microchip CE-SDS analysis was performed using a Lab-
on-chip microanalyser (Agilent). Free thiols were quanti-
fied using Ellman’s reagent. Metabolic analysis was con-
ducted by Metabolon (Durham, NC).
Results
Identification of disulphide reduction of IgG during
Primary Recovery
The IgG manufacturing process as transferred from the
co-developing partner included a cell culture settling
step following the Production Bioreactor and prior to
Primary Recovery.
Disulphide bond reduction was first detected during
initial development runs by routine Non-reduced (NR)
CE-SDS in-process analysis after Affinity capture chro-
matography (data not shown). NR-CE-SDS analysis
identified elevated levels of free light chain and half anti-
body molecules, when compared to Reference Standard.
Additional analysis, employing microchip-based NR-
CE-SDS methods indicated that the antibody reduction
occurred during the primary recovery cell settling step
* Correspondence: mullan_brian@lilly.com
1Manufacturing Science and Technology, Eli Lilly & Co, Kinsale, Cork, Ireland
Full list of author information is available at the end of the article
Mullan et al. BMC Proceedings 2011, 5(Suppl 8):P110
http://www.biomedcentral.com/1753-6561/5/S8/P110
© 2011 Mullan et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
(results not shown). This was confirmed by LC-MC ana-
lysis (results not shown). Assessment of disulphide
bonding pattern and intactness by LC-MS peptide map-
ping identified both inter- and intra-chain disulphide
scrambling (results not shown).
Delineation of events leading to IgG reduction
Initial investigations to understand process behaviour
during primary recovery identified that reducing species,
including free thiols (which increase over the course of
the Production Bioreactor, up to 1mM), were present at
the end of the Production Bioreactor (Figure 1a). Dis-
solved oxygen was also shown to deplete during the cell
settling phase following harvest (data not shown). From
this, an initial working hypothesis was formed that redu-
cing species, including free thiols, became reactive at
low dissolved oxygen concentrations and led to IgG1
disulphide bond reduction.
A revised process control strategy was implemented
(see below) to prevent oxygen depletion and maintain
dissolved oxygen levels above a minimum level. This
involved including an aerated and agitated hold for Clar-
ified Harvest Intermediate (CHI) in the process.
Further studies identified that O2 is critical to main-
taining a stable environment for oxidised (i.e., normally
disulphide bonded) IgG1 in CHI. When O2 was present,
IgG1 remained intact under all conditions evaluated.
Only when O2 was deliberately absent, or stripped away,
would the harvest material or CHI demonstrate poten-
tial for reduction (Figure 1b).
Metabolic behaviour of reducing intermediates
The working hypothesis was that by maintaining suffi-
cient levels of dissolved oxygen in the CFM, the thiol
species could be reacted out (oxidised) and a stable
environment for oxidised IgG1 created (Figure 1a). How-
ever, the relationship between IgG reduction and thiol
redox state is not first order (Figure 1c), and the rate of
thiol oxidation was found to be dependent on the
source of Production Bioreactor material (i.e., varied
with different harvest lots). This indicated the involve-
ment of an additional component, potentially catalytic,
which has not yet been identified in our studies. Thiore-
doxins have been identified as such a catalytic compo-
nent by others [5,6], and these need to be recycled after
one redox cycle via Thioredoxin Reductase / NADP(H).
Metabolic analysis of cell and media material from
Production Bioreactors indicated high levels of oxidised
homocysteine and cysteine (both reactive redox mole-
cules), which correlated with decreasing levels of folate
Figure 1 IgG disulphide bond reduction under various conditions for cell-settled and immediately clarified harvest material. CHI, Clarified harvest
intermediate; DiS, Disulphide; LoC, Lab-on-a-Chip (Agilent).
Mullan et al. BMC Proceedings 2011, 5(Suppl 8):P110
http://www.biomedcentral.com/1753-6561/5/S8/P110
Page 2 of 3
(B6) and cobalamine (B12), both of which are involved
in recycling homocysteine. Overall, this analysis identi-
fied numerous options for media optimisation to miti-
gate against IgG reduction. However, given the success
of process controls (described below), and the late stage
of process development (pre-validation) these media
optimisation options were not pursued.
Process controls to mitigate disulphide reduction of IgG
A process control strategy was implemented including:
• Establishing a minimal dissolved oxygen level in the
Production Bioreactor prior to harvesting
• Immediately clarifying the Production Bioreactor
material (i.e., eliminating the cell settling step)
• Holding the CHI in a hold vessel (LevMix container,
agitated hold) that had been partially pre-filled with pro-
cess air.
Conclusions
• Gross disulphide bond reduction was observed during
late stage development of an IgG1 monoclonal antibody
being commercialised for a therapeutic indication;
• Disulphide bond reduction had a second, or higher,
order link to low dissolved oxygen levels in process
intermediates, and the involvement of a catalytic factor
was also indicated;
• Implementation of an appropriate control strategy
(and associated process analytics) informed by process
development has ensured no recurrence of this issue
(for n=15 full scale lots).
Author details
1Manufacturing Science and Technology, Eli Lilly & Co, Kinsale, Cork, Ireland.
2Bioprocess R&D, Eli Lilly & Co, Indianapolis, Indiana, USA. 3Bioproduct
Analytical Chemistry, Eli Lilly & Co, Indianapolis, Indiana, USA. 4Analytical
Technical Operations, Eli Lilly & Co, Kinsale, Cork, Ireland.
Published: 22 November 2011
References
1. Wypych J, et al: Human IgG2 antibodies display disulfide-mediated
structural isoforms. J Biol Chem 2008, 283:16194-16205.
2. Liu YD, et al: Human IgG2 antibody disulfide rearrangement in vivo. J
Biol Chem 2008, 283:29266-29272.
3. van der Neut Kolfschoten M: Anti-inflammatory activity of human IgG4
antibodies by dynamic Fab arm exchange. Science 2007, 317:1554-1557.
4. Labrijn AF, et al: Therapeutic IgG4 antibodies engage in Fab-arm
exchange with endogenous human IgG4 in vivo. Nature Biotechnology
2009, 27:767-773.
5. Trexler-Schmidt M, et al: Identification and Prevention of Antibody
Disulfide Bond Reduction During Cell Culture Manufacturing.
Biotechnology and Bioengineering 2010, 106:452- 461.
6. Kao Y-H, et al: Mechanism of Antibody Reduction in Cell Culture
Production Processes. Biotechnology and Bioengineering 2010, 107:622-632.
7. Mullan B, et al: Transfer, Implementation and Late Stage Development of
an End-To-End Single-Use Process for Monoclonal Antibody
Manufacture. American Pharmaceutical Review 2011, 58-64.
doi:10.1186/1753-6561-5-S8-P110
Cite this article as: Mullan et al.: Disulphide bond reduction of a
therapeutic monoclonal antibody during cell culture manufacturing
operations. BMC Proceedings 2011 5(Suppl 8):P110.
Submit your next manuscript to BioMed Central
and take full advantage of: 
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at 
www.biomedcentral.com/submit
Mullan et al. BMC Proceedings 2011, 5(Suppl 8):P110
http://www.biomedcentral.com/1753-6561/5/S8/P110
Page 3 of 3


Disulphide bond reduction of a therapeutic monoclonal antibody during cell culture manufacturing operations

Crossref

BMC Proceedings

Mullan Brian

Dravis Bryan

Lim Amareth

Clarke Ambrose

Janes Susan

Lambooy Pete

Olson Don

O’Riordan Tomas

Ricart Bruce

Tulloch Alexander G

Directory of Open Access Journals

et al: Human IgG2 antibodies display disulfide-mediated structural isoforms.

et al: Human IgG2 antibody disulfide rearrangement in vivo.

et al: Mechanism of Antibody Reduction in Cell Culture Production Processes. Biotechnology and Bioengineering

et al: Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human

Identification and Prevention of Antibody Disulfide Bond Reduction During Cell Culture Manufacturing. Biotechnology and Bioengineering

Neut Kolfschoten M: Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange. Science

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3284901

Disulphide bond reduction of a therapeutic monoclonal antibody during cell culture manufacturing operations

Abstract

Similar works

Full text

Available Versions

Springer - Publisher Connector

Crossref

Directory of Open Access Journals