Download Validation of Viral Safety of Recombinant Proteins

Validation of Viral Safety of
Recombinant Proteins
Presented by Franz Nothelfer
DOWNSTREAM PROCESSING
C
heap, effective, and highly
focused tools for the elimination
of viruses from recombinant cells
are always much in demand
because of their wide spread use in the
production of biologic drugs. In this
presentation on viral safety aspects in
downstream development of recombinant
proteins, Nothelfer introduced the topic
by discussing some general rules for
comprehensive evaluation of this complex
issue. He stated that validation of virus
reduction should be performed before the
onset of clinical trials. At least two
orthogonal steps should be assessed. The
reproducibility of an effective virus
reduction step should be demonstrated
by at least two independent experiments.
Virus reduction studies should include
both an enveloped and a small
nonenveloped virus (preferably a
parvovirus). If cells containing endogenous
retroviruses or retrovirus-like particles are
used in the protocol, then a specific model
virus (such as a murine leukemia virus)
should be used to validate the
inactivation/removal of viruses to
demonstrate full clearance of particles
present in the unprocessed bulk.
In this context the term orthogonal, as
it relates to process development, is
generally understood to mean that
multistep purification procedures should
use separation mechanisms that are
distinct from one another.
Nothelfer discussed his team’s work
on different orthogonal steps used in the
downstream process for removing
viruses. One approach is the inactivation
Figure 1: Platform processes for monoclonal antibodies (MAbs); AEX = anion-exchange
chromatography; CEX = cation-exchange chromatography; UF/DF = ultra/diafiltration
Capture
Viral
Inactivation
Protein A
Chroma. 1
Capture:
removal of
proteins,
lipids, DNA
Depth
Filtration
AEX
CEX
Acid
Depth
AEX
Treatment Filtration Chroma. 2
CEX
Chroma. 3
Virus
inactivation
Removal
of
turbidities
Orthogonal viral inactivation/removal steps
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M arch 2017
Bulk
Bulk
Virus
Polishing:
drug
removal
removal of
substance
HCP, aggregates
Concentration,
adjusting
physical
Supportive viral removal steps
conditions
of enveloped viruses at low pH value or
through the use of detergents such as
Triton X100. An alternative line of attack
for the removal of small viruses is the use
of a filter with a 20-nm pore size.
Low pH treatment was found to be
extremely effective for removing
enveloped viruses in a model system. It
was determined that sodium acetate
buffer inactivates MuLV (murine leukemia
virus) completely at pH values <pH 3.9
and in sodium citrate buffer at a pH value
of 3.3. However, inactivation at higher pH
values was more difficult to predict for
the citrate buffer system.
Nothelfer noted the following
guidelines for recombinant protein
purification: The downstream process for
a recombinant protein should be able to
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15(3)s
Virus
UF/DF
Filtration
UF/DF
Downstream Platform Process for MAbs
Franz Nothelfer is associate director of protein science at
Boehringer Ingelheim Pharma, Germany.
28 BioProcess International
Viral
Filtration
eliminate more virus than is estimated to
be present in a single dose equivalent of
unprocessed bulk material. And low pH
treatment, virus filtration, and anionexchange chromatography are favored
because they represent robust virus
removal steps in a MAb purification
process.
It should be recognized that the
downstream treatment for a recombinant
protein should be able to eliminate
substantially more virus than is estimated
to be present in a single-dose equivalent
of unprocessed bulk. Before the
development of a downstream process of
a recombinant protein, the process steps
have to be evaluated as potential virus
removal steps.
It is essential that these guidelines be
followed precisely, because an
inadequate viral safety program may
cause a substantial delay of clinical
studies and approval of a marketable
product. For blockbuster biologic drugs,
that could result in losses running to
millions of euros per day.
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