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Improved Aggregate Reduction with Capto Adhere by Addition of Chaotropic Agents

IP.com Disclosure Number: IPCOM000187183D
Publication Date: 2009-Sep-01
Document File: 9 page(s) / 249K

Publishing Venue

The IP.com Prior Art Database

Abstract

A study on the effects of urea for reduction of Mab aggregates with Capto adhere has been performed. The results showed that by adding urea the monomer capacity could be decreased while the aggregate capacity was almost unaffected. This means that a higher yield was possible to achieve by adding urea into the load/wash buffer and, if present, the elution buffer. In this particular case the monomer yield was improved from 86% to 93%, a 7% unit increase, without affecting the HCP or aggregate reduction ability. In production situations this might be worth millions of dollars, since the price tag for monoclonal antibodies is very high and high dosage levels are used.

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Improved aggregate reduction with Capto adhere by addition of chaotropic agents

Summary

A study on the effects of urea for reduction of Mab aggregates with Capto adhere has been performed. The results showed that by adding urea the monomer capacity could be decreased while the aggregate capacity was almost unaffected. This means that a higher yield was possible to achieve by adding urea into the load/wash buffer and, if present, the elution buffer. In this particular case the monomer yield was improved from 86% to 93%, a 7% unit increase, without affecting the HCP or aggregate reduction ability. In production situations this might be worth millions of dollars, since the price tag for monoclonal antibodies is very high and high dosage levels are used.

1 Introduction

In monoclonal antibody (Mab) production one of the most important issues is the reduction of aggregated antibodies. Since the introduction to the market Capto adhere has been successfully used to reduce low or moderate amounts of aggregates from Mab preparations. However, if high amounts of aggregates are present (>10%) yield must often be sacrificed to be able to reach the purity target (usually <1% aggregate content). In this study the effect on the aggregate reduction of a chaotropic additive, such as urea, has been investigated.

1.1 Purpose and goal


The purpose was to get a better knowledge on how chaotropic agents affect the separation of aggregates from monomers when using Capto adhere. The goal was to be able to reduce high amounts of aggregates while maintaining a high monomer yield (>90%).

1.2 Chemical/technical Background


The interactions of Capto adhere are not fully known, but ion exchange, hydrophobic/aromatic interactions and hydrogen bonding are all involved, making the mechanistic understanding more difficult. One theory to be tested is if it possible to change the interactions between the Mab and Capto adhere by reducing the hydrogen bonding interactions, i.e. include a chaotropic agent such as urea in the solution.

1.3 Disposition of the study


The study has been set up as a high-throughput development (HTPD) study, i.e. PreDictor plates were used for screening and small columns for verification and scale up.

2 Experimental

2.1 Materials / Investigated units

2.1.1 Plates


PreDictor Capto adhere, 6 ┬Ál plates (GE Healthcare) were used for all the screening experiments.

2.1.2 Sample


A monoclonal antibody (Mab-2) expressed in a CHO cell line was used as sample. The antibody is prone to aggregation and aggregate levels of approximately 14% were present.

2.1.3 Buffers in the PreDictor plate studies


The buffers were prepared with a Tecan robotic system. The buffers were prepared by mixing different volumes of stock solutions and MilliQ. The stock solutions used for the different experiments are summarized in Table 1. All buffers were mixed at higher concentrations compared to the experiments since they were to be mixed...