Polishing Complex Therapeutic Proteins - A new downstream purification platform using a salt-tolerant membrane adsorber. - BioPharm International

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Polishing Complex Therapeutic Proteins
A new downstream purification platform using a salt-tolerant membrane adsorber.


BioPharm International
Volume 26, Issue 1, pp. 42-51

MATERIALS AND METHODS

Sartobind Q and Sartobind STIC membrane adsorbers in LP15 (0.41 mL) and nano (1 mL) formats were provided by Sartorius Stedim (Goettingen, Germany). The ligand for Sartobind STIC is a polyallylamine compared to the quaternary amine for Sartobind Q. The LP15 prototype is a 0.41 mL disk format device with three membrane layers in a polysulfone housing. The design of the LP15 device is similar to the commercially available syringe unit Sartobind MA15. Sartobind nano is the commercially available scale-down device with 15 layers and 36.4 cm2 total surface area. Scalability of Sartobind nano (1 mL) to process scale capsules has been well demonstrated through the entire range of product up to 1.62 L membrane. Cylindrical format, radial flow distribution, and bed height were kept constant to allow for linear scale up.

The recombinant protein feed materials for this study were obtained from clinical manufacturing at Bayer Berkeley, CA. The immunoaffinity columns used were directly scaled down from clinical manufacturing processes.

As in current processes, all purifications were carried out at 2–8 C to obtain maximum product stability. Step gradient experiments and breakthrough experiments were carried out on an AKTA Explorer 100 chromatography system from GE HealthCare (Uppsala, Sweden). Simple flow-through runs were carried out using a Watson Marlow 101U peristaltic pump (Falmouth, England). Initial development studies used Sartobind STIC LP15 devices at a flow rate of 3 mL/min (7.3 MV/min). Sartobind STIC nano was used for laboratory scale purifications of two different complex glycoproteins, Bay-A001 and Bay-A002, at a flow rate of approximately 5 mL/min (5 MV/min). The full factorial study on pH and conductivity was designed and data analyzed in JMP (SAS, Cary, NC). All buffers used in the anion exchange MA operation were imidazole based. No multivalent anion, such as phosphate or citrate, was used as it may interfere with protein adsorption because of its strong interaction with AEX ligands.

Bay-A001 and Bay-A002 have theoretical isoelectric points (pI) of 7.4 and 6.4, respectively, based on the amino acid sequences. However, due to the heterogeneity of post-translational modifications, the recombinant proteins exhibit high levels of heterogeneity in pI, which prevented pI determination using isoelectric focusing (IEF) electrophoresis.

All assays were performed by the analytical development and support group at Bayer, Berkeley, CA.


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