Comparative Study of Commercially Available Protein A Chromatography Resins and AmsphereTM A3

Sachiko Tsuda1; Takashi Tanaka2, Yuko Imanishi2, Kaori Itaya1; Takashi Ichii1;  Yusuke Okano1; Takashi Matsuda1; Masaaki Hanamura1

1 JSR Life Sciences Corporation — 25 Miyukigaoka, Tsukuba, Ibaraki, 305-0841, JAPAN  2 JSR Corporation — 25 Miyukigaoka, Tsukuba, Ibaraki, 305-0841, JAPAN

INTRODUCTION 

Host cell proteins (HCPs) in biopharmaceuticals must be controlled in the downstream process (DSP) of antibody production, since it has critical influence on product quality. Generally, the majority of HCPs in harvested cell culture fluid are removed during the affinity step using Protein A chromatography resin, and the remaining HCPs are remove in the polishing step by a combination of ion-exchange chromatography, hydrophobic interaction chromatography, etc. Thus, identification of remaining HCPs after Protein A affinity chromatography process can work as base information for fine establishment of DSP. In this study, HCPs in elution samples of Protein A affinity chromatography were evaluated with ELISA (quantitative analysis) and 2D-LC/MS (qualitative analysis). These results were multilaterally compared with JSR’s resin, Amsphere A3 and other commercially available resins.

EXPERIMENTAL CONDITIONS 

PURIFICATION

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Column (ID: 0.5 cm, BH: 1.0 cm), AKTA prime plus, GE Healthcare.

IMPURITY ANALYSIS 

• CHO Host Cell Protein ELISA Kit, 3rd Generation, F550, Cygnus Technologies.

• Quant-iTTM PicoGreen dsDNA Assay Kit, P11496, Thermo Fisher.

• TSKgel Super SW3000, 0.1 M PB, 0.1 M Na2SO4, pH 6.7, 0.35 mL/min, Tosoh.

2D-LS/MS ANALYSIS

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PROPERTIES OF PROTEIN A RESIN AND MODEL FEED 

Host cell proteins (HCPs) in biopharmaceuticals must be controlled in the downstream process (DSP) of antibody

Using two industrial monoclonal antibodies (mAbs) and an Fc-fusion protein, the performance of Amsphere A3 was compared to two of the most commonly used commercial Protein A resins.

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RESULTS 

CAPACITY AND IMPURITY CLEARANCE 

• Amsphere A3 exhibited higher DBC than both Agarose S/L.

• The impurity levels in the elution pools of Amsphere A3 were as low as Agarose S/L.

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IDENTIFICATION OF HCPS USING 2D-LC/MS 

• Most of the HCPs in the eluates of Amsphere A3 and Agarose S/L were in common.

• HCPs with pI < 6 were detected more than HCPs with pI > 8.

• It is interesting to note that HCPs with pI values between 6 and 8 were not detected regardless of the resin used.

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DISCUSSION 

2D-SDS/PAGE OF FEED 

The feeds for this study contain HCPs with pI from 3 to 10.

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CORRELATION BETWEEN AMOUNT OF IgG AND HCP (Trastuzumab BS)

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• No significant differences of the remaining HCPs are observed, although Amsphere A3 and Agarose S/L consists of different base matrix.

• From 2D-LC/MS analysis, pIs for the majority of remaining HCPs are less than 6. Taking into account of pI for target molecules, non-specific electrostatic interaction between HCPs and target molecule is considered to be a dominant factor in the residual mechanism of HCPs during Protein A chromatography.

• Quantitative HCP analysis showed linear correlation between IgG and HCPs amount, which indicates the direct interaction between IgG and HCPs.

• For Amsphere A3 and Agarose S/L, HCPs with pI in the pH range from 6 to 8 were not detected. Therefore, it is assumed that non-pecific hydrophobic interaction between HCPs and resin is well suppressed for both resins.

CONCLUSION 

• HCPs in elution samples of Protein A affinity chromatography were analyzed quantitatively and qualitatively.

• Amsphere A3 exhibits higher DBC and same level of step yield and impurity clearance as commercially avairable Agarose S/L.

• The results suggested that contaminated HCP species are mainly determined by electrostatic charge of HCP in washing steps.

• Amsphere A3 can replace the existing agarose-based Protein A affinity resins under the same binding/ wash conditions without specific improvement of subsequent polishing processes.

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