132 BioProcess International INDUSTRY YEARBOOK 2010 - 2011 ADVERTORIAL

I
nterferon beta-1b is a protein in the interferon family
used to treat the relapsing-remitting form of multiple
sclerosis (MS). Interferon beta-1b is a natural human
protein (molecular weight ~18,500 Da) that is produced in
the body in response to viral infection and has antiviral activity.
It has been shown to slow the advance of MS and reduce the
frequency of attacks. It is believed that interferon-beta achieves
this effect on MS progress via its anti-inflammatory properties.
Interferon beta-1b drugs have been approved for over 20 years
to treat the symptoms of MS. Interferon beta-1b is produced as
a recombinant protein in the bacterium E. coli by fermentation
and subsequent purification to the active drug. The E. coli
bacteria produce the interferon beta-1b molecule at low yield
and as an insoluble and inactive product. As part of the
purification process, the molecule must be restored to its active
state, a process known as refolding. Refolding a protein
molecule is a difficult, inefficient, and costly process. If
interferon beta-1b could be produced by a bacterium at high
yield in a fully active form without the necessity of refolding, it
would be much less expensive to produce and could be offered
to patients at a more affordable price.
The Pseudomonas fluorescens-based Pfnex Expression
Technology platform is a proven high-throughput, parallel
screening, protein expression strain development platform that
has been designed specifically to deliver bacterial production
strains expressing high yields of soluble and active protein. It is
based on an extensive toolbox of expression strategies that can
be seamlessly combined to deliver robust expression strains for
the production of recombinant proteins. Scientists at Pfnex
Inc. a San Diego, CAbased protein discovery, development,
and production company have cloned and expressed many
difficult-to-express proteins in their soluble and active forms
and successfully completed scale-up for manufacturing. Pfnex
scientists, using the platform, cloned the interferon beta-1b
coding gene into 20 unique expression plasmids and
transformed them into 30 phenotypically distinct P. fluorescens
host strains. The resulting 600 expression strains were grown in
96-well plates, and the expression of interferon beta-1b was
determined for each strain, all in about one month.
Subsequently identified, high-expressing strains were tested
in scaled-down fermentation vessels (at 4 mL and then 1 L
scales) using design-of-experiment tools under multiple
fermentation conditions. The results of these experiments
identified an interferon beta-1b production strain and
fermentation conditions yielding 8 g/L of soluble, active
interferon beta-1b. A downstream recovery and purification
process has been developed yielding highly purified and fully
characterized interferon beta-1b (Figure 1). This process is
cGMP-ready. Pfnex Inc. is now offering for sale reagent-grade
recombinant interferon beta-1b in small lots and seeking a
partner to develop the molecule for human therapeutic use.

Charles H. Squires, PhD, is vice president of discovery and

partnerships for Pfnex Inc., 5501 Oberlin Drive, San Diego, CA 92121;
1-858-352-4400, fax 1-858-352-4602; proteins@pfenex.com, www.
pfenex.com.
Human Interferon Beta-1b
Production in Pseudomonas fluorescens
Using the Pfnex Expression Technology Platform
Figure 1: LEFT, interferon beta-1b accumulation curves in four fermentations of P. fluorescens production strain, with maximum yields between >8 g/L
and 9 g/L; RIGHT, HPLC trace of purified interferon beta-1a, with specifications as shown in the inset
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Specication Result
Purity
Endotoxin
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>99% by SDS-CGE
<10 EU/mg
<100 ppm
As expected
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standard
Expression and Production