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interferon-beta-1b

Extraction and purification process for interferon-beta-1b

Fraunhofer Institute for Interfacial Engineering and Biotechnology

Therapeutic proteins continuously acquire higher shares of the pharmaceuticals market. Monoclonal antibodies, for example, show doubledigit growth rates and also proteins which have been known for a longer period of time, such as interferon- (IFN-) exhibit an annual turnover
of three billion US dollars. Treatment with IFN- is one of the current basic therapies for the most frequent form of multiple sclerosis (MS),
relapsing-remitting MS. The costs for IFN- treatment in Germany amount to 18,000 euros per patient and year with the consequence that the
majority of the 2.5 million MS patients worldwide cannot be adequately treated with IFN-. In this context, the required quantities of active
substances at 45 mg per patient and year for IFN--1b are very small.

Two forms of IFN-beta

There are currently two forms of IFN- on the market. The recombinant form prepared in animal cells (IFN--1a) corresponds to the human
form in amino acid sequence and glycosylation. In contrast, the commercially available recombinant variant expressed in E. coli is not
glycosylated and differs from the natural human form at its N-terminus (methionine is cleaved) and in that at position 17 cysteine has been
replaced by serine. The form prepared in E. coli is termed IFN--1b. The patent protection of the two forms is running out. As a result, the
opportunity of making IFN-b available as biosimilar to a broader group of patients more reasonably and in many parts of the world for the first
time ever has arisen.

Criteria for an optimized production process

The objective of a current project at Fraunhofer IGB is the development of a procedure for preparing IFN--1b on an industrial scale. In this
context, the price, the safety of the products, the stability of the processes (ruggedness), and the scalability are of central importance. A high
expression rate, low costs for media and fermentation, as well as simple and stable procedures for isolation and purification (downstream
processing) are decisive for the economic success and for safe patient care.

Cloning and expression

Scientists at Fraunhofer IGB have been able to establish a highly productive clone, which bears an IFN--1b gene sequence adapted to E. coli.
This allows the expression of the desired IFN--1b proteins within the cells as inclusion bodies. As a result of high-cell-density fermentation, a
stable and high expression rate of at least 20 percent IFN--1b in the total cell protein is achieved with this clone.

Scalable purification processes

Isolation, purification, and solubilization of the IFN- proteins from inclusion bodies have been a technically complex process up to now. The
objective was to work out a technically simple and scalable downstream process [DFW1] for IFN--1b. For this, the bacterial cells are disrupted
immediately after fermentation with a combined enzymatic-mechanical process. From this cell homogenate, the target protein is obtained with
high yield and selectivity in only one extraction step with 2-butanol. The hydrophobic interferon-b moves upward into the upper organic phase
(2-butanol) with the aid of surfactants. Bacterial proteins are found concentrated as a solid layer[DFW2] between the lower aqueous and upper
organic phase. The lower aqueous phase contains the hydrophilic substances of the cells such as salts and DNA/RNA. Figure 1 shows the
three phases after a phase separation forced by centrifugation. Figure 2 shows the protein pattern by means of SDS-PAGE before the
extraction step (lane 3) and in the organic phase (lane 4). The target protein is present with a fraction of 20 percent before the extraction step;
after extraction it has a purity of 95 percent. The following chromatographic steps increase the purity even further (Fig. 3).
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Fig. 1: A f ter centrif ugation of

the homogenates, three phases
f orm. The IFN--1b is located in
the upper, organic phase.

Fig. 2: SDS-PA GE gel

electrophoresis of the
homogenate bef ore extraction
(lane 3) and the organic phase
with IFN--1b (lane 4).

Fig. 3: SDS-PA GE gel

electrophoresis of IFN--1b
af ter f urther chromatographic
purif ication.

Perspective
With the technically simple and scalable purification process for IFN--1b established at the Fraunhofer IGB, the majority of the bacterial
proteins are separated directly in the first purification step. We can thus avoid the laborious isolation, purification and solubilization of the
inclusion bodies, which result in high losses. This process supplies IFN--1b in high purity, which can be reasonably manufactured on an
industrial scale.

Project partners
Cinnagen, Teheran, Iran
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Contact

Dr. Hans Weber

more info

Dr. Anke Burger-Kentischer

more info

Fraunhofer-Gesellschaft

Source: Fraunhofer-Gesellschaft: Fraunhofer Institute for Interfacial Engineering and Biotechnology - Extraction and
purification process for interferon-beta-1b
Online in the Web ; URLhttp://www.igb.fraunhofer.de/en/competences/molecular-biotechnology/therapeutic-proteins/purificationinterferon-beta.html
[Date: 24.06.2013, 14:41 hour]]

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