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High Resolution and High Efficiency Separations of mAbs and ADCs Using Proteomix HIC Columns

By: Stacy Shollenberger, Hillel Brandes, Reporter US Volume 33.4

Introduction

A variety of products derived from monoclonal antibodies (mAbs), including mAb fragments and antibody-drug conjugates (ADCs), are being developed for the treatment of cancer and other diseases due to their increased potency combined with reduced toxicity. However, the efficacy of these molecules is highly dependent upon the target site-specificity and binding properties of the mAb, the linker stability, the potency of the drug, and both the distribution and number of drug species on the mAb1. These requirements highlight the importance of characterizing these highly heterogeneous products using appropriate analytical techniques in order to assess and monitor them during manufacturing and subsequent storage.

Hydrophobic interaction chromatography (HIC) is a technique for protein separations and has been commonly used as an orthogonal method to size exclusion chromatography (SEC) and ion exchange (IEX) chromatography for the characterization of mAbs. Here we introduce Proteomix® HIC columns which have been designed for high resolution and high efficiency separations of proteins, oligonucleotides, and peptides.

General Description

Utilizing proprietary surface technologies, Proteomix HIC-NP resin is made of non-porous polystyrenedivinylbenzene (PS/DVB) beads with narrow-dispersed particle size distribution. As shown in Figure 1, the PS/DVB bead is modified with alkyl groups or an aryl group that provides hydrophobic interaction with analytes. Proteomix HIC-NP resin is highly rigid and mechanically stable. In comparison to silica based HIC phase media, Proteomix HIC-NP phases have advantages for biomolecule separations with wide pH range (2-12) and high thermal stability. The nonporous structure and narrow particle distribution offer special selectivity, high resolution separation of proteins such as mAbs, ADCs, and related protein fragments, as well as DNA and oligonucleotides. Proteomix HIC-NP media are applicable at laboratory discovery, laboratory-scale purification, and process chromatography for the production of a few mgs to kilogram of proteins.

Structure of Proteomix HIC-NP5 Resin

Figure 1. Structure of Proteomix HIC-NP5 Resin

Table 1. Technical Specifications

Resin Matrix: Spherical, highly cross-linked PS/DVB
Pore Size: Nonporous
Particle Size: 5 µm and 10 µm
Phase Structure: Ethyl, propyl, butyl, or phenyl
Separation Mechanism: Hydrophobic interaction (HIC)
pH Stability: 2-12
Operating Temperature: Up to 80 °C
Operating Pressure: Up to 6,000 psi
Mobile Phase Compatibility: Compatible with aqueous solution, a mixture of water and acetonitrile, acetone, methanol, or THF

Featured Characteristics

  • Highest capacity and resolution
  • Consistent lot-to-lot reproducibility
  • Improved protein recovery with intact biological activity
  • Negligible non-specific interactions
  • Ideal for separation and analysis of hydrophobic proteins, mAbs, and ADCs
  • Suitable for separation and analysis of general biological samples

High Stability and Lot-to-Lot Reproducibility

Proteomix HIC columns are based on PS/DVB resin and all the surface coatings are chemically bonded onto PS/DVB support, which provides exceptionally high stability. The columns are compatible with most aqueous buffers, such as ammonium sulfate, sodium acetate, phosphate, Tris, and a mixture of water and acetone, methanol, acetonitrile and THF. When 25 mM sodium phosphate buffer, at pH 7.0, was used as the mobile phase to run the Proteomix HIC Butyl-NP5 column, 400 injections or 3 months of usage has negligible deterioration of the column.

Proteomix HIC columns provide high lot-to-lot consistency on ADC, mAb, and protein separations as shown in Figures 2-4.

Proteomix HIC Butyl-NP5 for Herceptin-cysteine ADC Separation-Lot Consistency Testing

Figure 2. Proteomix HIC Butyl-NP5 for Herceptin-cysteine ADC Separation-Lot Consistency Testing

Conditions
column:
Proteomix HIC Butyl-NP5, 4.6 × 35 mm, 5 µm (Product No. 61864-U); mobile phase: [A] 2 M ammonium sulfate in 0.025 M sodium phosphate, pH 7.0; [B] 0.025 M sodium phosphate, pH 7.0; [C] 100% IPA; flow rate: 0.8 mL/min; column temp.: 25 °C; detector: UV, 214 nm; injection: 10 µL; sample: ADC, 1 mg/mL in 1M ammonium sulfate

 

Proteomix HIC Butyl-NP5 for Herceptin-mAb Separation - Lot Consistency Testing

Figure 3. Proteomix HIC Butyl-NP5 for Herceptin-mAb Separation - Lot Consistency Testing

Conditions
column:
Proteomix HIC Butyl-NP5, 4.6 × 35 mm, 5 µm (Product No. 61864-U); mobile phase: [A] 2 M ammonium sulfate in 0.025 M sodium phosphate, pH 7.0; [B] 0.025 M sodium phosphate, pH 7.0; [C] 100% IPA; flow rate: 0.8 mL/min; column temp.: 25 °C; detector: UV, 214 nm; injection: 10 µL; sample: herceptin, 1 mg/mL in 1 M ammonium sulfate

 

Proteomix HIC Butyl-NP5 for Protein Separation - Lot Consistency Testing

Figure 4. Proteomix HIC Butyl-NP5 for Protein Separation - Lot Consistency Testing

Conditions
column:
Proteomix HIC Butyl-NP5, 4.6 × 35 mm, 5 µm (Product No. 61864-U); mobile phase: [A] 2 M ammonium sulfate in 0.1 M sodium phosphate, pH 7.0; [B] 0.1 M phosphate, pH 7.0; flow rate: 0.4 mL/min; column temp.: 25 °C; detector: UV, 214 nm; injection: 4µL; sample: ovalbumin 1.0 mg/mL, Chymotrypsinogen 0.5 mg/mL

Additional Applications

In Figure 5, the Proteomix HIC Butyl-NP5 column was used for the characterization of the distribution of drug-linked species and the determination of the average drug to antibody ratio (DAR) after peak integration. Because HIC separates molecules based on their hydrophobicity, the Proteomix HIC Butyl-NP5 column is very effective for this type of separation due to the fact that hydrophobicity increases with the number of attached payloads.

Herceptin and its ADCs Separation on Proteomix HIC Butyl-NP5 Column

Figure 5. Herceptin and its ADCs Separation on Proteomix HIC Butyl-NP5 Column

Conditions
column:
Proteomix HIC Butyl-NP5, 4.6 × 35 mm, 5 µm (Product No. 61864-U); mobile phase: [A] 2 M ammonium sulfate in 0.025 M sodium phosphate, pH 7.0; [B] 0.025 M sodium phosphate, pH 7.0; [C] 100% IPA; flow rate: 0.8 mL/min; column temp.: 25 °C; detector: UV, 214 nm; injection: 10 µL; sample: herceptin/ADC1/ADC2, 1 mg/mL in 25 mM sodium phosphate

Legal Information

Proteomix is a registered trademark of Sepax Technologies

Materials

     

 References

  1. Wu, A. M.; Senter, P. D. Arming Antibodies: Prospects and Challenges for Immunoconjugates. Nat. Biotechnol. 2005, 23(9), 1137-1146.

 

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