Process for the industrial-scale purification of gamma globulins from human plasma for industrial applications
Inventors
Nardini, Claudia • Morelli, Andrea • Farina, Claudio • Esposito, Sabrina • Lazzarotti, Alessandra • Petrucci, Arianna
Assignees
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Publication Number
US-8933204-B2
Publication Date
2015-01-13
Expiration Date
Abstract
The invention relates to a novel, industrial-scale process for the purification of gamma-immunoglobulins (IgG) starting from plasma or fractions thereof. The method involves two chromatographic steps, i.e. a cation exchange capture chromatography, and then a polishing anion exchange chromatography, ensuring a highly purified end product, which contains no aggregates, and high yields. The process also involves a virus inactivation step by means of a solvent/detergent treatment to inactivate the viruses with a lipid envelope, and a virus removal step by nanofiltering to ensure the removal of the non-enveloped viruses.
Core Innovation
The invention relates to a process for the production of IVIG using human plasma or an intermediate plasma fraction enriched in IgG as starting material. The process includes sequential chromatographic steps in which IgG is first captured on a strong cation exchanger and then polished on a strong anion exchanger. In the capture step, the strong cation exchanger has SO3− functional groups, and in the polishing step, the strong anion exchanger has quaternary ammonium groups as exchanger groups.
Viral safety is addressed by performing at least one viral inactivation step between the two chromatographic steps using a solvent-detergent treatment, and by subjecting the solution containing IVIG to a viral inactivation step by nanofiltration after the second chromatographic step. The process further specifies conditioning, charging, washing, and elution conditions for both chromatographic resins, including buffer, ionic-strength, and pH-based requirements.
The approach is described in the context of industrial-scale production of IVIG with attention to yield and purity characteristics. It is also presented with structured resin-loading and elution conditions for the strong cation exchanger and the strong anion exchanger.
Claims Coverage
The document contains one independent claim covering an IVIG production process based on sequential capture on a strong cation exchanger (SO3− groups) and polishing on a strong anion exchanger (quaternary ammonium groups), with solvent-detergent viral inactivation between steps and nanofiltration viral inactivation after polishing. The claim also includes specified resin loading and buffer, ionic-strength, and pH conditions.
Sequential IgG capture and polishing on strong cation and anion exchangers
A process for production of IVIG with sequential chromatographic steps comprising an IgG capture step on a strong cation exchanger and an IgG polishing step on a strong anion exchanger, where the strong cation exchanger has SO3− functional group and the strong anion exchanger has quaternary ammonium groups as exchanger groups.
Solvent-detergent viral inactivation between chromatographic steps
At least one viral inactivation step is performed between the first chromatographic capture step and the second chromatographic polishing step by means of a solvent-detergent treatment.
Nanofiltration viral inactivation after polishing
After the second chromatographic step, the solution containing IVIG is subjected to a viral inactivation step by means of nanofiltration.
Defined cation-exchange conditioning and salt elution
For the strong cation exchanger: charging is performed with 20–60 mg of IgG/ml of resin, conditioning, charging, and washing are performed using a 25–375 mM buffer at pH 5.4–5.7, and elution is performed using a buffer having 0.25–1 M sodium chloride concentration.
Defined anion-exchange conditioning and selective elution by ionic strength or pH variation
For the strong anion exchanger: charging is performed with 20–60 mg of protein/ml of resin, conditioning, charging, and washing are performed using a 10–330 mM buffer at pH 9.0–10.5 with NaCl 8–10 mM, and elution is performed using increased ionic strength or by means of pH variation, with corresponding conductibility and pH ranges specified for each elution mode.
Overall, the claim coverage centers on a two-step sequential chromatography for IVIG, combined with solvent-detergent viral inactivation between steps and nanofiltration viral inactivation after polishing, and further constrained by specified resin loading and buffer, ionic-strength, and pH conditions.
Stated Advantages
High yield and high purity, including low aggregates/degradation products.
Improved scalability versus prior precipitation-based and multi-step chromatographic approaches.
Avoidance of no preliminary lipoprotein removal.
Reduced/optional ethanol use (for fraction I precipitation only).
Documented Applications
Industrial-scale production of IVIG (intravenous immunoglobulin) from human plasma or an IgG-enriched intermediate plasma fraction.
Use of Cohn fraction I cryosupernatant/supernatant as starting material for the IVIG production process.
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