Redefining the Interface
Between Upstream and
Downstream Processing
Pete Gagnon, Hui Theng Gan, Lihan Tan, Wei Zhang, Rui Nian
Bioprocessing Technology Institute, Singapore
BioProcessing Asia, Langkawi, Malaysia, November 3-6, 2014
Introduction
One of the most basic principles of process development is
that any given purification method should be preceded by a
step to remove agents that would interfere with the ability of
the method to fulfill its intended role.
The need to remove solids is obvious, but stopping there
represents an assumption that interference is mediated
exclusively through physical clogging.
Recent studies show that chemical interference also
depresses fractionation performance, severely at capture,
and significantly during later polishing steps.
Luhrs et al, J. Chromatogr. B 877 (2009) 1543. • Mechetner et al, J. Chromatogr. B 879 (2011) 2583.
Gagnon et al, J. Chromatogr. A 1218 (2011) 2405. • Gan et al, J. Chromatogr. A 1291 (2013) 33.
Gagnon et al, J. Chromatogr. A 1324 (2014) 171. • Gagnon et al, J. Chromatogr. A 1340 (2014) 68.
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Introduction
All data in this presentation were obtained with an IgG1
monoclonal antibody immunospecific for human epidermal
growth factor receptor 2, produced at up to 2.8 g/L by ~1011
CHO cells/mL in protein-free media, from a tri-cistronic
vector developed at BTI.
Ho et al, J. Biotechnol. 157 (2012) 130.
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The chemical interface
Chromatin expelled from dead host cells is a major contributor
to aggregate formation and source of host cell contamination.
Individual nucleosomes contain 147 bp of DNA wrapped 1.65 times around a core
octamer of histones ((H2A,H2B,H3,H4)2). DNA and histones each contribute about
100 kDa. Nucleosomes are linked in linear arrays by 20-80 bp linker DNA segments
associated with histone H1.
Image modified from scbt.sastra.edu
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The chemical interface
Intact nucleosomal arrays persist in cell culture harvest
Fractions from SEC in 2 M NaCl, agarose electrophoresis
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The chemical interface
Native size distribution of major contaminant classes
SEC, harvest clarified by centrifugation-microfiltration
Nucleosome arrays provide the structural core of chromatin heteroaggregates
that range in size from about 50-700 nm. DNA and histones dominate their
chemical behavior, but 80% of their mass is from non-histone proteins.
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The chemical interface
Size distribution of host proteins, rSDS-PAGE of SEC fxns
HMW fxns contain more species than all others, most <100 kDa.
Histone identities confirmed by Mass Spec and Western blots.
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The chemical interface
Why has the role of chromatin not been recognized?
Broad spectrum HCP immunoassays do not detect histones.
Accurate estimation of histone content requires dedicated
histone assays.
Even dedicated histone assays require up to 16 hour sample
extraction at pH values below 1, and some require antibody
addition to the sample under those conditions.
Accurate determination of total HCP requires that broad
spectrum and dedicated histone assays be run, and the
results added together.
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The chemical interface
Why has the role of chromatin not been recognized?
DNA also requires severe sample preparation: up to 16
hours exposure to proteinase K in 0.5% SDS at 50°C.
Lacking such preparation, DNA quantitation and
determination of size distribution are both confounded.
DNA association with chromatin also points to another
analytical challenge:
All of the DNA in cell culture harvests is histone-associated.
It behaves differently than purified DNA. Are clearance
results from spike-recovery models with purified DNA valid?
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The chemical interface
How does chromatin respond to different conditions?
Under physiological conditions:
Strong binding of DNA to histones retards its lysis by
endogenous nucleases released at cell death.
Inter-nucleosomal linker DNA becomes apparent first, then
lysis of core DNA releases histone octamers.
Liberated core octamers dissociate stepwise to hexamers,
tetramers, dimers, then individual histones.
Liberated histones bind secondarily to exposed DNA on the
perimeter of still-intact nucleosomes.
Secondary associations of non-histone HCP with chromatin
remain fairly stable.
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The chemical interface
How does chromatin respond to different conditions?
In 2 M NaCl:
DNA dissociates from histone core octamers.
Histone core octamers are stabilized and form aggregates.
Liberated core octamers co-aggregate with histones
released from no-longer-existing nucleosomes.
Secondary associations between chromatin and nonhistone proteins are destabilized.
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The chemical interface
How does chromatin respond to different conditions?
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The chemical interface
Take-home:
Composition of chromatin heteroaggregates varies substantially
as a function of buffer conditions.
That means their composition varies across different
fractionation environments – different kinds of chromatography.
It also means their composition varies across the stages of a
chromatography method, and across processes.
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Interactions with chromatography media
Protein A as a model
Under loading conditions, the nucleosomal cores of most
heteroaggregates are insulated by surrounding non-histone
proteins.
Their affinity for protein A is nil, and these heteroaggregates are
eliminated in the flow-through.
Heteroaggregates with exposed histone elements bind strongly
to protein A carboxyl groups.
At 50-700 nm, they are mostly too large to enter particle pores,
so they accumulate on the surface where they interfere with the
entrance of IgG into the pores.
This depresses binding capacity.
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Interactions with chromatography media
Protein A as a model
Under elution conditions, histone-protein A contacts and some
intra-heteroaggregate associations persist, but others are
destabilized and become dissociated.
Dissociated contaminant subsets include individual species and
heteroaggregates. These leachates are mostly soluble under
elution conditions, but become turbid upon neutralization.
Note: that means turbidity of protein A eluates is not an indicator
of low pH-mediated damage to the antibody.
Heteroaggregate components that remain bound after elution
are mostly removed by 4 M guanidine, and more effectively by
50 mM NaOH, but complete removal requires 500 mM NaOH.
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Interactions with chromatography media
The overall pattern is the same with cation exchange and
electronegative multimodals, but the contaminant subsets that
flow-through, that are bound, or leach during elution, or remain
bound, vary with the chemistry of the solid phase and with the
elution conditions.
The pattern is conceptually similar with electropositive
multimodals applied in bind-elute mode, except that binding is
mediated through DNA.
The leaching factor is eliminated with polishing methods
applied in flow-through mode because there is no change of
conditions to dissociate contaminant subsets from strongly
bound heteroaggregates.
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Interactions with chromatography media
CHA: chromatin heteroaggregate. 1-3) Bound CHA restricts pore access. 4) compound accumulation of CHA. 5) CHA without external
histone contact flow through the column. 6,8) histone-dominant CHA remnants restrict pore egress. 7) individual non-histone dissociated
from CHA by elution conditions contaminants eluted IgG. 9) CHA dissociated from compound association with still-retained CHA also
contaminates eluted IgG.
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Managing chromatin
The most effective approach is to remove chromatin in advance.
Removal can target the DNA component or the histone component.
Management tools:
Ethacridine: intercalates and destabilizes DNA, weakens interactions
between DNA and histones, co-precipitates proteins.
Caprylic acid: co-precipitates proteins by accumulating on their
electropositive domains. Very high affinity for histones.
Allantoin: co-precipitates aggregates though hydrogen bonding.
Solid phases: particles, membranes, monoliths, depth filters, etc.,
with chromatin-complementary surface chemistry.
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Managing chromatin
Comparison of harvest clarified by different methods.
Redux of/clar.met.
Cent.MicF
Ethacridine
Caprylate
Host DNA
nil
5-6 logs
5-6 logs
Histone host proteins
nil
> 95%
> 99%
Non-histone host proteins
nil
60-90%
90-99%
Aggregates > 300 kDa
nil
99%
99%
Aggregates < 300 kDa
nil
90-99%
90-99%
IgG fragments
nil
0-50%
50-95%
MVM
nil
4 logs
5 logs
MuLV
nil
5 logs
9 logs
Final turbidity
10-50 NTU
2 NTU
2 NTU
IgG recovery
90-95%
98-99%
90-95%
Ethacridine and Caprylate refer to hybrid methods that include solid phase
adsorbents to enhance performance.
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Managing chromatin
Analytical SEC after different clarification methods
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Benefits of managing chromatin
Protein A, traditional clarification vs advance chromatin removal
The contaminants in IgG eluted from protein A are not bound to IgG, and
they do not co-elute with IgG.
They dissociate from more strongly bound chromatin heteroaggregates at
the same time IgG is dissociated from its interaction with protein A.
Advance chromatin removal suspends this contamination pathway.
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Benefits of managing chromatin
Protein A, traditional clarification vs pre-elution wash
Pre-elution washes pre-leach contaminant subsets from strongly retained
chromatin heteroaggregates.
This reduces the mass of contaminants that can leach during elution of IgG,
but leachable contaminants remain.
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Benefits of managing chromatin
Protein A, purification summary, washes vs advance removal
Param/Met.
Traditional
Pre-E wash
AdvChromX
Capacity
25 mg/mL
25 mg/mL
31 mg/mL
DNA
13 ppm
6 ppm
2 ppm
HCP
2,006 ppm
708 ppm
< 1 ppm
Turbidity
108 NTU
26 NTU
6 NTU
The ability of pre-elution washes to improve purification performance is fractional
compared to advance chromatin removal, they provide no improvement in dynamic
binding capacity, and they do not reduce cumulative fouling of the adsorbent.
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Benefits of managing chromatin
Eshmuno™ HCX, traditional clarification vs advance chromatin removal
Retained chromatin heteroaggregates obstruct pores and reduce IgG capacity
more than 3-fold. They also interfere with elution.
More than 95% of the contaminants in the IgG fraction are leached from
chromatin heteroaggregates that remain bound under elution conditions.
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Benefits of managing chromatin
Purification summary: Clar. > Eshmuno HCX > Capto™ adhere
Step/Clar.
Traditional
AdvChromX
OM
DNA
His
Nhis
4,722 ppm
28,359 ppm
377,976 ppm
4 ppm
LOQ
27,302 ppm
> 1,000x
> 28,000x
14x
HCX
Capacity
DNA
His
Nhis
29 mg/mL
65 ppm
1,474 ppm
125,507 ppm
94 mg/mL
4 ppm
LOQ
3,704 ppm
3x
16x
>1,400x
34x
CA
Capacity
DNA
His
Nhis
Aggr
9 mg/mL
60 ppb
LOQ
2,682 ppm
0.05%
11 mg/mL
3 ppb
LOQ
77 ppm
0.05%
20%
20x
-----35x
------
Net recov.
71%
86%
17%
His: histone host proteins. Nhis: non-histone host proteins. Aggr: aggregates
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Improvement
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Benefits of managing chromatin
Chromatin-directed clarification enables many capture options
and a reduction of processing steps.
Method
HCP
aggregates
Protein A > Capto adhere
< 1 ppm
< 0.05%
Cation exchange > Capto adhere
< 1 ppm
< 0.05%
Cation exchange > hydroxyapatite
< 1 ppm
< 0.05%
Multimodal (HCX) > VEAX
< 1 ppm
< 0.05%
VEAX > Capto adhere
< 1 ppm
< 0.05%
SXC > Capto adhere
< 1 ppm
< 0.05%
VEAX: anion exchange chromatography in void exclusion mode.
Nian et al, J. Chromatogr. A. 1282 (2013) 127-132.
SXC: steric exclusion chromatography with magnetic nanoparticles.
Gagnon et al, J. Chromatography A 1324 (2014) 171.
All HCP values with Cygnus Generation III HCP ELISA.
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Conclusions
Chromatin is a keystone variable and defining element of the
interface between upstream and downstream processing.
It plays a role in aggregate formation and it influences the
behavior of hundreds of host contaminant species.
Its composition and chromatographic behavior are strongly
influenced by the local chemical environment.
It depresses capacity and fractionation performance of all
purification methods.
Its extreme physicochemical features make it a challenge for
analytical methods.
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Conclusions
Awareness of chromatin’s bioprocessing impact provides a
starting point for managing it.
Integrating chromatin characterization into upstream operations
could lead to cell culture processes that enhance performance
and reproducibility of DSP.
Characterizing chromatin during DSP development leads to
more effective and better understood purification processes.
These include many alternatives to the traditional protein A
platform – many with only two chromatography steps.
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Cautions
Any recombinant protein produced in cells and harvested
from preparations that contain dead cells will contain
chromatin or its analogs.
Even yeasts and bacteria require DNA-compaction proteins
to organize their genetic material, and those proteins are all
referred to as histones or histone-like.
The issue extends to viruses, since their production typically
kills the cells that host them.
Whether recognized or not, chromatin is an important
process variable for all biological product classes.
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EndNotes
If you know the enemy and know yourself, you need not
fear the result of a hundred battles.
–Sun Tzu
Thanks to Sarah Maria Abdul Latiff, Jeremy Lee, Chiew Ling Lim, Cindy
Chuah, Dawn Goh, Phyllicia Toh, Xinhui Wang, Qiaohui Yang, Aina Hoi,
Denise Leong, Amrita Rai, Edmund Lim, Xuezhi Bi, Cheong Nge, and
Jimmy Chao for performing experiments and analyses; Yuan Sheng Yang,
Han Ping Loh, Su Jun Low, and Jake Chng for providing cell culture
media. Thanks also to Validated Biosystems, to the Biomedical Research
Council of the Singapore Agency for Science, Technology, and Research,
and to Exploit Technologies Pte. Ltd. for supporting this work.
Information/reprints: [email protected]
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