Separation of interleukins by a preparative chromatofocusing-like method

Area of Science:

• Biochemistry
• Protein Chemistry
• Chromatography

Background:

• Recombinant protein production often yields variants like deamidated forms.
• Accurate separation of these variants is crucial for therapeutic protein quality control.
• Existing methods for separating deamidated proteins can be complex and difficult to scale.

Purpose of the Study:

• To describe a novel chromatofocusing-like method for large-scale separation of deamidated from amidated recombinant human interleukin-1 alpha.
• To demonstrate the method's applicability to other recombinant proteins, such as interleukin-1 beta.

Main Methods:

• High-performance liquid chromatography (HPLC) with a sulfopropyl strong cation-exchange column.
• Utilized a gradient separation without carrier ampholytes or high ionic strength for elution.
• N-Terminal sequence analysis to confirm protein identity and deamidation site.

Main Results:

• Successfully separated two major protein species of recombinant human interleukin-1 alpha with distinct isoelectric points (pI 5.3 and 5.1).
• Identified the pI 5.1 species as the deamidated form, with Asp at position 32 instead of Asn.
• Demonstrated the method's effectiveness for separating N-Met from des-Met recombinant human interleukin-1 beta.

Conclusions:

• The developed chromatofocusing-like method is effective for large-scale purification of deamidated recombinant proteins.
• This technique offers an alternative to standard chromatofocusing and ion-exchange chromatography.
• The method is versatile and applicable to the separation of other protein variants.