Process for the purification of recombinant human il-11

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The invention relates to process for purification of recombinant human IL-1 1 from microbial cells. The method involves purification using hydrophobic interaction chromatography and ion exchange chromatography. The method further comprises use one or more fusion tags which provides solubility to the protein and also simplifies the purification of the target protein.

The invention relates to process for purification of recombinant human IL-1 1 from a soluble IL-1 1 fusion protein expressed in bacterial system. The method involves purification of the soluble fusion protein, digestion to separate the IL- 1 1 from the fusion tag followed by hydrophobic interaction chromatography and ion exchange chromatography.

The development of techniques and methods for protein purification has been an essential prerequisite for many of the advancements made in biotechnology. Protein purification varies from simple one step precipitation procedures to large scale validated production processes. The key to successful and efficient protein purification is to select the most appropriate techniques, optimize their performance to suit the requirements and combine them in a logical way to maximize yield and minimize the number of steps required. Most commonly used purification methods include use of affinity tags, fusion tags, metal binding, immunoaffinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography and HPLC. Recombinant DNA developments over the past decade have revolutionized the production of proteins in large quantities. While production of heterologous proteins in bacterial hosts has been implemented successfully in the biotechnology industry, there are numerous instances where bacterial expression systems have given less than satisfactory results. Often, over expression leads to the production of inclusion bodies, that are insoluble aggregates of misfolded proteins. For example, when high-expression levels are achieved, recombinant proteins are frequently expressed in Escherichia coli as insoluble protein aggregates termed "inclusion bodies" that have been the subject of many protein folding studies. The conditions for refolding the denatured protein must be optimized for each specific protein, and the renaturation yield may be low even in an optimized system. Thus it is often desirable to maximize the expression of the protein in a completely soluble form. US 5215895 relates to a novel cytokine that stimulates the function of cells of the immune and hematopoietic systems, and to processes for obtaining the factor and producing it by recombinant genetic engineering techniques. It describes expression of IL- l l in bacterial cells where the purification involves four ion exchange steps and cleavage of the fusion protein by hydroxylamine.

In one general aspect the invention is related to a method for purification of recombinant human Interleukin- 1 1 from bacterial cells, the process comprising the steps of:

(a) purifying the fusion protein using hydrophobic interaction chromatography,

(b) cleaving the fusion protein with enterokinase, and

(c) purifying IL-1 1 by ion exchange chromatography, wherein the purification steps may be in any order.

In another aspect of the invention the hydrophobic interaction chromatography is performed using resins selected from butyl sepharose, phenyl sepharose or octyl sepharose. In another aspect of the invention the Ion exchange chromatography is selected from cation exchange or anion exchange or both in either order.

In yet another aspect of the invention the method for purification of recombinant human Interleukin 1 1 comprises use of one or more fusion tags selected from SD tag, GM tag, T7 tag, GST tag, His tag, Trx Tag or MBP tag. The SD tag used in the invention having sequence Id 1 , comprises of 49 times repeated, Serine Aspartate residues. The fusion tag (having a pi of 4.0) has a total of 107 amino acids.

The details of one or more embodiments of the inventions are set forth in the description below. Other features, objects and advantages of the inventions will be apparent from the description and claims.