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POLISHING OF MONOCLONAL ANTIBODIES POST PROTEIN A AFFINITY CHROMATOGRAPHY

IP.com Disclosure Number: IPCOM000195912D
Publication Date: 2010-May-21
Document File: 4 page(s) / 156K

Publishing Venue

The IP.com Prior Art Database

Abstract

Anion exchange based methods have become, due to their well documented ability to clear virus, DNA, endotoxins, and many protein impurities, an industry standard for large scale polishing of monoclonal antibodies. The most common formats used for polishing of antibodies are beaded particles or membranes derivatized with quaternary amines. In this paper we have studied the capacity to remove host cell proteins (HCP) and DNA at relatively short residence times of 1 minute or lower on quaternary amine based plain agarose resins and dextran grafted agarose resins. The dextran grafted resin showed a more than 10 fold increase in DNA and HCP removal capacity compared to the plain agarose resins. This paper presents results of HCP and DNA removal capacity under different process conditions.

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Polishing of monoclonal antibodies post Protein A affinity chromatography

Materials

Q SepharoseTM Fast Flow: A plain agarose based strong anion exchanger

CaptoTM Q: A dextran grafted agarose based strong anion exchanger

Membrane A: A regenerated cellulose based Q membrane, 5.0 cm2

Membrane B: A polyethersulfone based Q membrane, 4.9 cm2

OH Base matrix

Agarose
90 µm (average)

N

+

OH

+ + N

+

Base matrix
High flow agarose 90 µm (average)

Surface extender Quaternary amine (Q-group) Animal-free dextran

Experimental

DNA capacity: Sonicated salmon sperm DNA dissolved to a concentration of 0.1 mg/ml in 100 mM sodium phosphate buffer, pH 6.5.

Dynamic capacity at 10 % break through (QB10) was determined by frontal analysis at different residence times.

Host cell protein (HCP) capacity: A Protein A purified monoclonal antibody preparation was, after conditioning to the respective buffer condition, used for frontal analysis.

MAb concentration: 8.9 - 9.4 mg/ml
HCP concentration: 2500 - 3000 ng/ml
Buffer pH 6.4, 4 mS, 25 mM sodium phosphate, 19 mM sodium chloride.

Buffer pH 7.4, 10 mS, 28 mM phosphate, 73 mM sodium chloride

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Host Cell Protein Capacity

Membrane A Q Sepharose FF

Capto Q

Q Sepharose FF

pH 6.4, 4 mS cm-1, R 1.0 min

250

250

00.0 2.0

pH 6.4, 4 mS

         cm-1, Rt 0.4 min

200

200

00.0 2.0 4.0 6.0 Load (kg.L-1)

HCP (ppm)

HCP (ppm)

150

150

100

100

50

50

         4.0 6.0 Load (kg

. L-1 )

pH 7.4, 10 mS cm

-1, Rt 0.4 min

pH 7.4,10 mS cm-1, Rt 1.0 min

250

00.0 2.0 4.0 6.0 Load (kg

.L-1)

250

00.0 2.0 4.0 6.0 Load (kg . L-1)

200

200

HCP (ppm)

HCP (ppm)

150

150

100

100

50

50

Fig.1 Frontal analysis under two different conditions, pH 7.4, 10 mS/cm and pH 6.4, 4 mS/cm, at two different residence times, 0.4 and 1.0 minutes. The sample had a MAb concentration of 9 mg/ml and a HCP content of 2500-3000 ng/ml.

Process economy and productivity

In several publications (1,2) membranes are compared to a resin developed more than 25 years ago, Q Sepharose Fast Flow. However there are a number of new resins available with significantly improved pressure/flow properties allowing linear flow velocities of more than 1000 cm/hour. In ref. 1, a process economy calculation is based on the assumption of a process load of 70 g/l resin at a linear flow velocity of 200 cm/h versus a process load of 10700 g/l membrane at a linear velocity of 600 cm/h. However, as we have shown the HCP and DNA clearance capacities of the dextran grafted resin are within the same range as for the membranes, even at linear...