Analysis of Monoclonal Antibody and Related Substances using a New Hydrophobic Interaction Chromatography (HIC) Column

Transcription

1 Analysis of Monoclonal Antibody and Related Substances using a New Hydrophobic Interaction Chromatography (HIC) Column Julia Baek, 1 Robert van Ling, 2 Xiaodong Liu 1 1 Thermo Fisher Scientific, Sunnyvale, CA, USA; 2 Thermo Fisher Scientific, Breda, The Netherlands

2 Overview Purpose: Demonstrate high resolution separation of mabs and mab related substances using Thermo Scientific TM MAbPac TM HIC-1 column. Methods: Ammonium sulfate and sodium phosphate mobile phase were used. In some cases,addition of isopropanol to both mobile phases increased the resolution of the chromatogram. Results: The new MAbPac HIC-1 column was developed using advanced surface bonding technology to achieve unique selectivity, high recovery and high efficiency. High resolution separation of proteins and various mab samples were successfully carried out using a MAbPac HIC-1 column. Introduction Monoclonal antibodies (MAbs) are the most prominent class ofprotein therapeutics because of their high specificity, excellent biocompatibility and effectiveness against autoimmune disorders, cardiovascular diseases, infectious diseases, and cancer. The proliferation of monoclonal antibody therapeutics and their susceptibility to various biochemical modifications has highlighted the importance of characterizing these highly heterogeneous products for their safety and efficacy. Hydrophobic interaction chromatography (HIC) is a technique for separation of proteins including monoclonal antibodies. The HIC mobile phase usually consists of a salting-out agent, which at high salt concentration, retains the protein by increasing hydrophobic interaction between the protein and the stationary phase. HIC has been widely used as an orthogonal method to size exclusion chromatography and ion exchange chromatography for analysis of mab and related substances, such as succinimides, fragments, oxidants, C-terminal lysine modification, and drug conjugates, to monitor the stability and potency of the drug. 2 Analysis of Monoclonal Antibody and Related Substances using a New Hydrophobic Interaction Chromatography (HIC) Column

3 Here we introduce a new HIC column designed for mab analysis. It is based on high-purity, spherical, wide-pore (1, Å), 5 µm silica particles functionalized with proprietary alkyl amide groups. The advanced surface bonding technology provides desired selectivity, excellent recovery, high efficiency, and chemical stability. Its effectiveness for mab separation has been demonstrated by examples including mab fragments, oxidized mab, PEGylated mab, mab aggregates and antibody-drug conjugates (ADCs). Methods Samples Monoclonal antibody samples and ADC sample were donated by biotech companies. Proteins and other chemicals were from Sigma-Aldrich. Column MAbPac HIC-1, 5 µm, mm (P/N 8848) Liquid Chromatography HPLC experiments were carried out using Thermo Scientific Dionex UltiMate 3 BioRS system equipped with: SR-3 Solvent Rack (PN ) LPG-34RS Biocompatible Quaternary Rapid Separation Pump (PN 54.36) WPS-3TBRS Biocompatible Rapid Separation Thermostatted Autosampler (PN ) TCC-3RS Rapid Separation Thermostatted Column Compartment (PN 573.) VWD-34RS Rapid Separation Variable Wavelength Detector (VWD) equipped with micro flow cell (PN 574.1) Chromatography was controlled by Thermo Scientific Dionex Chromeleon Chromatography Data System. Method Development Ammonium sulfate and sodium phosphate based mobile phases were used. For the applications with mab and mab related substances, mobile phases were optimized by either lowering the starting salt concentration or adding isopropanol into both mobile phase A and mobile phase B. Thermo Scientific Poster Note PN2124 ISC 814S 3

4 FIGURE 1. Fast separation of standard protein mixture Column: MAbPac HIC-1, 5 µm Format: mm Mobile phase A: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. Mobile phase B: 1 mm sodium phosphate, ph Temperature: 3 ºC Flow rate: 1. ml/min Inj. volume: 1 µl Detection: UV (28 nm) Sample: Protein mixture Peaks: 1) Myoglobin 2) Ribonuclease A 3) Lysozyme 4) α-chymotrypsinogen A Analysis of mab Fragments Analysis of antibody fragments is important for both characterization of Fab or Fc based biotherapeutics and localization of the sources of heterogeneities on a monoclonal antibody molecule. HIC can provide the resolution required for the separation of Fab and Fc fragments and their hydrophilic or hydrophobic variants. 1 Figure 2 shows a comparison of an intact mab and its papain digest on MAbPac HIC-1. The MAbPac HIC-1 column efficiently separates Fab and Fc fragments and further separates variants of these fragments. These variant peaks imply oxidation or other modifications in these fragments. FIGURE 2. Separation of papain digested mab a b F ab Intact mab F c Column: MAbPac HIC-1, 5 µm Format: mm Mobile phase A: 1.5 M ammonium sulfate, 5 mm sodium phosphate, ph 7. / isopropanol (95:5 v/v) Mobile phase B: 5 mm sodium phosphate, ph 7. / isopropanol (8:2 v/v) Temperature: 25 ºC Flow rate: 1. ml/min Inj. volume: Papain digest:1 µl (1 mg/ml) Intact mab: 2 µl (5 mg/ml) Detection: UV (28 nm) Sample: a) Intact mab b) Papain digest Analysis of Monoclonal Antibody and Related Substances using a New Hydrophobic Interaction Chromatography (HIC) Column

5 Analysis of Oxidized mab Monoclonal antibodies are susceptible to oxidation during storage and delivery. Oxidation of methionine or tryptophan residues have been linked to decreased or loss of bioactivity of mab therapeutics. 2 Therefore it is critical to monitor oxidation to confirm the stability and clinical efficacy of mab products. MAbPac HIC-1 column was able to separate oxidized mab from the native mab as shown in Figure 3. FIGURE 3. Separation of oxidized mab Column: MAbPac HIC-1, 5 µm Format: mm Mobile phase A: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. Mobile phase B: 1 mm sodium phosphate, ph Temperature: 3 ºC Flow rate: 1. ml/min Inj. volume: mab: 2 µl (2.5 mg/ml) Oxidized mab: 4 µl (1.3 mg/ml) Detection: UV (28 nm) Sample: mab Oxidized mab Analysis PEGylated mab PEGylated proteins are an important class of modern biotherapeutics. In general, PEGylation decreases immunogenicity, increases circulatory time and reduces proteolysis, resulting in enhanced medical efficacy. 3 Typically it is desirable to attach one PEG at a specific site. However since the reaction is not always site specific and more than one PEG may be attached it is important to purify the PEGylated protein and analyze the purity afterwards. Two derivatives of PEGylated mabs were successfully separated using MAbPac HIC-1 column in 2 minutes (Figure 4). Lowering the starting ammonium sulfate concentration to.6 M by simply changing the gradient resulted in a higher resolution separation. Thermo Scientific Poster Note PN2124 ISC 814S 5

6 FIGURE 4. Separation of PEGylated mabs Column: MAbPac HIC-1, 5 µm Format: mm Mobile phase A: 1.5 M ammonium sulfate, 5 mm sodium phosphate, ph 7. Mobile phase B: 5 mm sodium phosphate, ph Temperature: 3 ºC Flow rate:.5 ml/min Inj. volume: 1 µl (1 mg/ml) Detection: UV (28 nm) Sample: PEGylated mab Peaks: 1) Derivative 1 2) Derivative Analysis of mab Aggregates Protein and antibody aggregates are formed either during product expression in cell culture, downstream processing or storage. These aggregates may cause undesirable immune reactions which affect the safety of the drug. SEC is the most widely used technique for the detection and quantification of protein aggregates in biological drug products. However, several researchers have reported the use of HIC for the removal of protein aggregates. 4,5 Figure 5 demonstrates the separation of monoclonal antibody aggregates from the monomer form on the MAbPac HIC-1 column. In HIC, aggregates typically elute later than the main peak due to the increased hydrophobicity. FIGURE 5. Separation of mab aggregates Aggregate Column: MAbPac HIC-1, 5 µm Format: mm Mobile phase A: 1.5 M ammonium sulfate, 5 mm sodium phosphate, ph 7. / isopropanol (95:5 v/v) Mobile phase B: 5 mm sodium phosphate, ph 7. / isopropanol (8:2 v/v) Temperature: 25 ºC Flow rate: 1. ml/min Inj. volume: 5 µl (8 mg/ml) Detection: UV (28 nm) Sample: Monoclonal antibody Analysis of Monoclonal Antibody and Related Substances using a New Hydrophobic Interaction Chromatography (HIC) Column

7 Analysis of Antibody-Drug Conjugate Mimic Antibody drug conjugates (ADCs) are a rapidly growing class of protein therapeutics that target cancer cells. 6 Hydrophobic interaction chromatography is often used for the separation of ADCs since attachment of cytotoxin alters the hydrophobicity of the antibody. The least hydrophobic unconjugated antibody elutes first and as the number of attached drugs increases the elution time of each ADC increases as well. Therefore, HIC is often used to characterize the distribution of ADC molecules with different drug-to-antibody ratios (DARs). Figure 6 shows the separation of a cysteine-conjugated ADC mimic sample on the MAbPac HIC-1 column. The ADC mimics were conjugates between a drug mimic and mab via the sulfhydryl group of interchain cysteine residues which results in a mixture of drugloaded antibody species with to 8 drugs (Figure 6). The unmodified mab and ADCs with DAR values ranging from 2 to 8 are well resolved by the MAbPac HIC-1 column. The best peak shape and separation were achieved using 5% IPA in mobile phase A and 2% IPA in mobile phase B at 25ºC FIGURE 6. Separation of CYS-conjugated ADC mimic Column: MAbPac HIC-1, 5 µm Format: mm Mobile phase A: 1.5 M ammonium sulfate, 5 mm sodium phosphate, ph 7. / isopropanol (95:5 v/v) Mobile phase B: 5 mm sodium phosphate, ph 7. / isopropanol (8:2 v/v) Temperature: 25 ºC Flow rate:.5 ml/min Inj. volume: 5 µl (5 mg/ml) Detection: UV (28 nm) Sample: Cys-conjugated ADC mimic Peaks: 1) Unconjugated mab 2) DAR 2 3) DAR 4 4) DAR 6 5) DAR 6 6) DAR 8 Thermo Scientific Poster Note PN2124 ISC 814S 7

8 Conclusion The new MAbPac HIC-1 column was developed using wide pore silica and advanced surface bonding technology to achieve unique selectivity, high recovery and high efficiency. Fast and high resolution separation of proteins was achieved using MAbPac HIC-1 column. High resolution separation of various mab samples including mab fragments, oxidized mab, PEGylated mab, mab aggregates and ADC mimic were obtained using MAbPac HIC-1. References 1. Valliere-Douglass, J. et al. J.Chromatogr. A (28) 1214, Pan H. et al. Protein Sci. (29) 18, Veronese, F.M. and Mero, A. Biodrugs (28) 22, McCue JT et al. Bioprocess Biosyst Eng. (28) 31, Lu Y. et al. Curr Pharm Biotechnol. (29)1: Leavy, O. Nat.Rev. Immunol.(21) 1, Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Africa Australia Austria Belgium Brazil Canada China (free call domestic) Denmark Europe-Other Finland France Germany India Italy Japan Korea Latin America Middle East Netherlands New Zealand Norway Russia/CIS Singapore Sweden Switzerland Taiwan UK/Ireland USA PN2124-EN 716S