Polishing host cell proteins with HCPure™ mixed-mode adsorbent by astreabioseparations

Polishing host cell proteins with HCPure™ mixed-mode adsorbent Caroline Daye, Ian Scanlon, Koldo Morante Astrea Bioseparations, Horizon Park, Barton Road, Comberton, Cambridge, CB23 7AJ, UK

Abstract

The use of Protein A adsorbents to capture monoclonal antibodies is a routine process step. However, to reach the purity required, intermediate and polishing steps are often employed in multistep purification processes. Here we demonstrate clearance of host cell impurities with a novel adsorbent comprising a proprietary mixedmode ligand on beaded agarose, HCPure™. Primary capture of monoclonal antibodies and fragments was achieved with the GORE® Protein Capture Device with Protein A, a composite PTFE membrane with a native Protein A ligand. The use of intermediate washes with high salt, low pH, or arginine were investigated for optimizing host cell protein removal on the GORE® Protein Capture Device. A stringent wash regime was demonstrated to recover pressure increases to allow more than 20 cycles on the same device. Post-affinity mAbs and fragments were then polished using HCPure™, to remove host cell impurities such as host cell proteins (HCP) and DNA (HC DNA), aggregates, Protein A leachate, viruses, and endotoxin. HCPure™ adsorbent was highly effective for post-Protein A polishing of an IgG produced in CHO and HEK293 cells.

Exploring the pH wash regime for Protein A capture

Clarified CHO IgG was loaded on to a 1 mL GORE® Protein Capture Device with Protein A equilibrated with PBS. The device was washed with PBS followed by a high salt wash of PBS + 1.8 M NaCl. The salt was washed off with PBS before being eluted with 100 mM sodium citrate pH 3.4. As a comparison to this, an identical run was performed with a 100 mM sodium citrate pH 5 wash added prior to the elution. The run with the pH 5 wash had a similar impurity profile but a lower IgG yield. IgG recovery (mg)

IgG recovery (%)

Aggregate (%)

HCP concentration (ng/mg IgG)

Control

185

With pH 5 wash

184

HCPure™ has been shown effective for post-affinity polishing of full-length IgG, IgG fragments, and Bispecific IgG from CHO, HEK293, E. coli, and Pichia cell lines. The optimal running conditions for HCPure™ relies on the host cell and the target protein. A screen of pH (4 – 9) and conductivities (6 – 18 mS/cm) should be performed for each target.

HC DNA concentration (ng/mg IgG)

IgG concentration was measured by analytical Protein A HPLC, aggregates by SEC HPLC, host cell protein (HCP) by ELISA and HC DNA by PicoGreen.

50 mg of IgG from a neutralized Protein A elution fraction was loaded directly on to a 1 mL HCPure™ prepacked column in flow through mode. The column was equilibrated and washed with 80 mM sodium citrate, 170 mM Tris pH 6, 12 mS/cm to match the buffer conditions of the neutralized Protein A elution. The IgG was recovered in the flow through.

An additional batch of CHO IgG was purified with two further runs on a 9 mL GORE® Protein Capture Device. One of the runs eluted the IgG immediately after the PBS post-load wash, while the other was run with a high salt wash, followed by an arginine wash prior to the elution.

The IgG recovery was 96% and the HCP was reduced from 380 PPM to 50 PPM, within the FDA recommendation.

Three times less host cell protein impurities were eluted with the high salt and arginine washes, while still demonstrating minimal impact on IgG yield. Comparing HCP content post Protein A with high salt and arginine washes

Buffer

Equilibration

PBS

Load

CHO harvest

1000

PBS + 1.8 M NaCl

EQ wash

PBS

Arginine wash

PBS + 0.1 M arginine pH 7.5

Elution

100 mM sodium citrate pH 3.4

Strip

0.1 M citric acid

CIP

0.1 M NaOH

(mg or ppm)

PBS

High salt wash

600 400 200 0

IgG recovered (mg)

HCP (ppm)

No intermediate washes

High salt and arginine washes

Flow rate recovery of GORE® Protein Capture Device during cleaning

465

652

559

Polishing of HEK293 IgG using HCPure™ adsorbent

An additional mAb produced in HEK293 cells was purified with a GORE® Protein Capture Device followed by polishing with HCPure™ adsorbent. The column was equilibrated with 40 mM sodium phosphate, 160 mM NaCl pH 6, 18 mS/ cm. The Protein A elution fraction was dialysed into the equilibration buffer then 30 mg loaded on to a 1 mL HCPure™ pre-packed column and the IgG collected in the non-bound. The IgG recovery was 80% and had reduction of HCP, dsDNA, Protein A leachate and endotoxin impurities.

Impurity removal from HEK IgG with HCPure™ 250 200 150 100 50 0 IgG (mg)

HCP (PPM)

dsDNA (PPM)

Protein A (PPM)

Endotoxin (EU/mg)

Post-HCPure™

Any data or results provided are only examples and do not provide any guarantee of similar results in future. The products of Astrea Bioseparations may be covered by or for use under one or more patents: astreabioseparations.com/patents All trademarks, trade names, trade dress, product names and logos are the property of Astrea UK Services Ltd or their respective owners.

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(1) GORE® cleaning regime

PBS

Cle

3.6

0.2 M NaOH

Post-HCPure™

HCPure™ adsorbent is a mixed-mode chromatography resin which can be used for polishing of post-Protein A purified products. Reduction of host cell proteins, host cell DNA, Protein A leachate and endotoxin from IgG produced in CHO and HEK293 cells lines is shown. By utilizing a unique chemical space compared to other commercially available adsorbents, HCPure™ offers a valuable addition to a purification toolbox.

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200 mM arginine pH 2.3

Post-Protein A

Summary

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(10-minute hold)

Protein A leachate (ppb)

IgG concentration was measured by UV, host cell protein (HCP) and Protein A leachate by ELISA, HC DNA by PicoGreen and endotoxin by LAL assay

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Flow rate (mL/min)

50 mM Tris, 100 mM NaCl, 1% SDS, 10 mM DTT pH 10.4

HC DNA concentration (ppb)

Extended cleaning method recovers flow rate on GORE® Protein A device

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Buffer

HCP concentration (ppb)

Load

The same 9 mL device was used for more than 20 cycles of CHO IgG feedstock. The flow rate automatically reduced to maintain the pressure below the limit (0.4 MPa), indicating an increasing of pressure. The flow rate reduced in subsequent cycles but was recovered after the extensive cleaning, indicated by the teal bars below.

200 mM arginine pH 2.3

Aggregate (%)

800

IgG concentration was measured by UV280 and host cell protein (HCP) by ELISA

IgG recovery (mg)

IgG concentration was measured by UV, aggregates by SEC HPLC, host cell protein (HCP) and Protein A leachate by ELISA and HC DNA by PicoGreen.

1200

Post-load wash

Polishing of CHO IgG using HCPure™ adsorbent

This Protein A purified CHO IgG had relatively low impurities, but the host cell protein (HCP) concentration is above the FDA recommended 100 PPM (2). Astrea Bioseparations’ HCPure™ adsorbent was used as a polishing step following the Protein A device.

Exploring an arginine wash regime for Protein A primary capture

Step

Chemical space diagram of commercial mixed-mode adsorbents

(mg, ppm or EU/mg)

Polishing host cell impurities with HCPure™ adsorbent

HCPure™ is a mixed-mode adsorbent which has a unique chemical space compared to other commercially available adsorbents. HCPure™ primarily utilizes hydrogen bonding and hydrophobic interaction chromatography to bind host cell impurities whilst leaving the target protein in the non-bound.

(1) GORE® application note: Cleaning protocols to reduce the effects of pressure rise over the lifetime of a GORE® Protein Capture Device (2) doi: 10.1080/19420862.2021.1955811 HCPure™ Adsorbent is available at astreabioseparations.com