This document discusses challenges in applying standard platform purification processes to Fc-fusion proteins. Fc-fusion proteins show more diversity than monoclonal antibodies, which can complicate rapid process development. Residue levels and yields after protein A capture can vary significantly depending on cell line and molecule properties. Alternative viral inactivation or protein A wash steps may be required. Anion exchange chromatography may require bind-and-elute mode rather than flow-through for some molecules. Ultrafiltration/diafiltration can cause unexpected pH offsets at low protein concentrations for some Fc-fusions due to Donnan and volume exclusion effects. Overcoming these challenges may require modified purification steps or formulation conditions.

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ACS 2019
BIOT Section
Application and adaptation of
platform and alternative
purification steps to Fc-fusion
proteins
Nathan Nicholes Ph.D.
Scientist II, DSPD

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Fc-Fusion Protein Diversity
Platform Development Strategies
Platform Adaptations to address purification challenges
-resHCP clearance
-Aggregation during low pH VI
-UF/DF low concentration pH offsets
Conclusions
Technical Presentation Outline

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Fc-Fusion proteins are a significantly more diverse class of molecules than mAbs or
bispecific antibodies
Hydrophobicity, pI, Charge Distribution, Glycosylation etc.
Application of Platform-Based Expedited DSPD approach to enable rapid IND filing entails
some risks due to greater molecule diversity
Process development anticipated to utilize platform conditions for ProA,
low pH VI, VF and UFDF.
Development and optimization anticipated for polishing steps
This presentation will highlight differences between Fc-Fusion proteins that fit platform
purification steps well vs. those that did not and alternative purification strategies for more
challenging molecules.
Fc-Fusion Proteins and Platform DSPD Development

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Platform relies on ProA for capture from harvest
Low pH VI, Virus Filtration and UF/DF are expected
to require minimal optimization
Platform polishing steps rely on pI of molecule being
significantly different from most resHCP (pI > 7.0)
Optimization and robustness evaluation expected for
polishing chromatography steps
mAb/Fc-Fusion Platform Purification Evaluation
Viral Inactivation
AEX Based Polishing Step
CEX Based Polishing Step
Virus Filtration
ProA Capture
Ultrafiltration/Diafiltration

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This presentation will discuss specific challenges that
we commonly encounter in adapting platform
purification steps to more unusual Fc-fusion proteins
-resHCP removal over capture and VI
-Detergent Viral Inactivation
-AEX Gradient vs. Isocratic Elution Development
-UF/DF Low concentration pH offsets
mAb/Fc-Fusion Platform Purification Development Challenges
Viral Inactivation
AEX Based Polishing Step
CEX Based Polishing Step
Virus Filtration
ProA Capture
Ultrafiltration/Diafiltration

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resHCP levels post capture can vary widely from molecule to molecule
As a CDMO, we see a variety of different cell lines that vary in both
productivity and purity
Typical range for ProA eluate is 1000 – 20,000 ppm resHCP
ProA eluate resHCP levels setup clearance requirements for subsequent
steps
resHCP clearance achieved in platform purification through
AEX or AEX/MMC polishing step
Post load modulator washes on ProA
Redundant clearance on CEX or second polishing step
Platform process resHCP clearance

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Lower resHCP levels can be
cleared with more common
arginine-based intermediate
washes
More aggressive ProA washes
can remove more resHCP
however they also lower yields
and can impact product quality
Yield losses occur as product is
washed from the column during
the modulator wash phase
resHCP and Yields for two Fc-fusions with different ProA modulators

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Platform purification process employs low pH viral inactivation post capture
Low pH viral inactivation requires that the product be stable at pH conditions
that are low enough to inactivate enveloped viruses
Product instability at low pH is a more frequent problem with Fc-fusion proteins
and requires implementation of alternative viral inactivation strategy
Detergent Viral Inactivation can be applied pre or post ProA capture
Application before ProA capture typically requires larger volumes of
detergent but also reduces detergent levels in subsequent process
steps
Viral Inactivation Development

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resHCP levels were
significantly lower with
detergent viral inactivation of
the harvest material
resHCP clearance is likely due
to detergent disrupting
product/HCP interactions on
the column
Step yields were similar
despite the presence of
detergent in harvest material
Impact of Detergent Viral Inactivation on resHCP levels and yields post ProA

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AEX is a critical part of the platform purification process for Viral Clearance as
well as removal of resDNA and resHCP
Platform mAb/Fc-fusion AEX purification typically operates in flow through mode
and can achieve high product loading on the resin (50 - 300 g/L)
It is not always possible to operate at a pH that will allow for AEX operation in
flowthrough for acidic Fc-fusion proteins and achieve a robust separation
between product and resHCP
Column loading capacities are significantly lower for AEX in bind and
elute mode
AEX resin selection and chromatography development

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Isocratic elution is preferred due to lower operational risk and simpler process
characterization for late phase process development
For early phase clinical supply, however, gradient elution can provide both higher yields
and better resHCP clearance relative to isocratic elution conditions.
AEX Elution Mode (Isocratic vs. Gradient)

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Platform UFDF conditions assume that buffer exchange occurs without
selective partitioning of ions across the membrane
Gibbs-Donnan equilibrium effects are typically observed at high protein
concentrations for mAbs (>100 g/L)
We have observed that some Fc-fusion proteins show pH offsets
between the diafiltration buffer and the final formulated BDS at
significantly lower concentrations (≤50 g/L)
UFDF Development Challenges with Fc-Fusion Proteins

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“…when nondiffusible charged molecules, such as proteins, are
participating in an equilibrium process such as UF/DF, the nonideality of
the process, due to the unequal partitioning of charged solutes
across the membrane, yields an unequal distribution of
electrolytes and therefore results in significant differences in molar
buffer and excipient concentrations between the final formulation and
the DF buffer. This phenomenon has been attributed to the Donnan
and volume exclusion effect.”
Abel J et al. J Pharm Sci 107:1296-1303, 2018
UFDF Development Challenges with Fc-Fusion Proteins

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“The Donnan effect can cause a large offset in pH from the target value
established with the diafiltration buffer during the concentration and
diafiltration of charged proteins with ultrafiltration membranes. For
neutral formulations, the pH will typically increase above the diafiltration
buffer pH for basic monoclonal antibodies and decline below the
diafiltration buffer pH for acidic Fc-fusion proteins.”
From Bolton GR et al. Biotechnol. Prog., 27(1):140-152, 2011
UFDF Development Challenges with Fc-Fusion Proteins

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Certain formulation buffer/molecule pairings result in pH offsets between the
diafiltration buffer and the final formulated material
Options for addressing these offsets include
Evaluate product stability at pH values around the pH of the final
formulated BDS
Increasing buffer ion strength
Assessing pH offset with diafiltration buffer
Redeveloping formulation to a formulation system that does not show
an offset (not always an option)
UFDF pH offsets at low protein concentrations

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Diversity of Fc-fusion proteins can complicate the application of platform purification
strategies for rapid process development
Detergent inactivation in harvest provided additional, unanticipated reductions in resHCP in
the ProA eluate pool
Gradient elution on AEX can provide higher yields and better product quality relative to
isocratic elution conditions although it does entail some operational risk
Some Fc-fusion proteins can show unusual behavior during UF/DF at lower concentrations
that requires modification of the TFF operation or formulation buffer
Conclusions

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Questions?
Nate Nicholes
Nnicholes@kbibiopharma.com
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