Monoclonal Antibodies and Biosimilars

Monoclonal Antibodies and Biosimilars

Monoclonal antibodies (mAbs) have emerged as revolutionary therapeutic agents for an array of human diseases. In addition to their large mass and complex structure, mAbs are varied in their origin, makeup, effector function and delivery, and therefore require thorough formulation development, including characterization, quantitation and preservation.

More and more special designed Antibody drug conjugates (ADCs) are under development as targeted therapy for treating cancer. ADCs combine the targeting capabilities of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs, allowing new “proximity” based dosing strategies.

As therapeutic development moves from IgG-based antibodies to technologies like bispecific antibodies, peptibodies, and fusion proteins, we are leading innovation in analytical techniques. nSMOL (nano-surface and molecular orientation limited proteolysis) is Shimadzu’s novel approach that enables selective proteolysis of the Fab region of monoclonal antibodies. The nSMOL Antibody BA Kit is a ready-to-use reagent kit for collecting monoclonal antibodies from blood or other biological samples using immunoglobulin collection resin, and then performing selective proteolysis of the Fab region of these antibodies via trypsin-immobilized nanoparticles. Variable region-derived peptides produced by limited proteolysis can then be quantified via MRM measurements utilizing a high-performance liquid chromatograph mass spectrometer.

As first and second generation antibody therapeutics migrate off patent, there is a need to provide the same robust analysis to biosimilar therapeutics. The need to characterize not only the sequence of expressed mAbs, but the various post-translational modifications (PTMs) that can occur is key to proving bioequivalence for regulatory filings. Solutions for N-linked glycan analysis, charge variant analysis, and aggregation state are all available from your partners at Shimadzu.

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Featured LC-MS Applications

Monoclonal Antibody Workflows on the LCMS-9030

Monoclonal Antibody Workflows on the LCMS-9030

The Shimadzu Q-TOF LCMS-9030 plus Protein Metrics software offers a complete workflow solution for monoclonal antibody characterization. Customizable workflows can be created for a type of analysis including intact data, subunit, peptide fragments and deglycosylation analysis.

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N-Linked Glycan Analysis of Monoclonal Antibodies, Biosimilars, and Glycoproteins with High Resolution Mass Spectrometry and Fluorescence Detection

N-Linked Glycan Analysis of Monoclonal Antibodies, Biosimilars, and Glycoproteins with High Resolution Mass Spectrometry and Fluorescence Detection

The Shimadzu LCMS-9030 has shown excellent mass accuracy for N-linked glycans on several proteins. The Restek Raptor Polar X, an innovative hybrid ion-exchange/HILIC column, is an ideal column for glycans as reflected by the baseline separation of the structurally similar N-glycans. Protein Metrics Glycan Workflow offers additional workflows to other published methods.

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LCMS Bioanalysis of Antibody Drugs Using Fab-selective Proteolysis "nSMOL Method"

LCMS Bioanalysis of Antibody Drugs Using Fab-selective Proteolysis "nSMOL Method"

This report describes a selection protocol of signature peptides suitable for pharmacokinetic studies.

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In-depth Peptide Mapping of Monoclonal Antibody (mAb) by A de novo Peptide Sequencing Method on Q-TOF Mass Spectrometer with Data-Independent Acquistion

In-depth Peptide Mapping of Monoclonal Antibody (mAb) by A de novo Peptide Sequencing Method on Q-TOF Mass Spectrometer with Data-Independent Acquistion

In this report, we demonstrated an integrated MS full-scan and MS/MS Diag approach on Shimadzu LCMS-9030 (Q-TOF) mass spectrometer for de novo peptide sequencing of mAb.

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Disulfide Bond Characterization of Monoclonal Antibody (mAb) using Q-TOF Mass Spectrometer

Disulfide Bond Characterization of Monoclonal Antibody (mAb) using Q-TOF Mass Spectrometer

A  straightforward LCMS-based method for accurate disulfide bond peptide characterization of mAb biosimilar was established on LCMS-9030 (Q-TOF).The MS/MS spectra with fragmentation data provide high confidence results on sequencing analysis.The demonstrated performance for bevacizumab biosimilar in detection and de novo sequencing of non-reduced and reduced disulfide bonds signifies its practicability for the structural characterization of mAb biosimilars.

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Featured MALDI Applications

A Study on a Method for Evaluating Glycans in Biopharmaceuticals

A Study on a Method for Evaluating Glycans in Biopharmaceuticals

In this article, we report the results of studying a method for releasing O-glycans chemically in which the peeling reaction is suppressed, based on a PMP labeling method.

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A Study on a Method for Evaluating Glycans in Biopharmaceuticals - Part 2

A Study on a Method for Evaluating Glycans in Biopharmaceuticals - Part 2

This article introduces the results of comparing some of the pretreatment methods widely used in N-glycan analysis and investigating how they affect the analysis results.

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Characterization of Glycan Binding Site of O-Linked Glycopeptides Using MALDI-7090 High Resolution MALDI-TOF MS

Characterization of Glycan Binding Site of O-Linked Glycopeptides Using MALDI-7090 High Resolution MALDI-TOF MS

In this article, we present a method for determining the O-linked glycan binding site that uses partial digestion with multiple enzymes and the MALDI-7090 high-resolution MALDI-TOF MS.

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Simplified Mass Measurement of Chemically-Modified Antibodies: Determination of the Presence of the Number of Modifications Using a Linear Benchtop MALDI-TOF MS

Simplified Mass Measurement of Chemically-Modified Antibodies: Determination of the Presence of the Number of Modifications Using a Linear Benchtop MALDI-TOF MS

This article introduces an example of analyzing the pseudo ADC, which was created by artificially binding low-molecular compounds to a standard research antibody, using a benchtop MALDI-TOF MS.

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Affinity Purification of IdeZ digest for Glycosylation Profile of Immunoglobins using a linear benchtop MALDI-TOFMS

Affinity Purification of IdeZ digest for Glycosylation Profile of Immunoglobulins using a linear benchtop MALDI-TOFMS

Glycosylation on protein plays wide-range vital roles in biological processes from stabilization of protein conformation to expression of binding specificity. A characterization of the N-/O-linked glycan is quite significant, especially in development of biopharmaceuticals. To date, whereas intensive efforts were conducted to characterize glycans precisely with high-end mass spectrometers, conventional instruments without time consuming preparation have been anticipated for batch analysis in screening or QA/QC. 

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Analysis of Modification Site of Chemically Modified Antibody Using MALDI-Mini-1 Compact MALDI DIT Mass Spectrometer

Analysis of Modification Site of Chemically Modified Antibody Using MALDI-Mini-1 Compact MALDI DIT Mass Spectrometer

Antibody drug conjugates (ADC) are a new class of anti-cancer drugs in which an antibody is bound to a cytotoxic drug.  Because they combine the high substrate specificity of the antibody and the effect of a low-molecular drug,  ADC are expected to be more effective anti-cancer drugs than the conventional low-molecular drugs. When a different compound is bound artificially to a protein, the binding degree of that compound and its binding site become one of the critical quality properties. A pseudo ADC was created by artificially binding a low-molecular compound to a standard research antibody, and was then analyzed using a MALDImini-1.

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Analysis of N-Linked Glycan using MALDI-Mini-1 Compact MALDI Digital Ion Trap Mass Spectrometer: Structural Analysis and Identification of Sialyl Linkage Isomers

Analysis of N-Linked Glycan using MALDI-Mini-1 Compact MALDI Digital Ion Trap Mass Spectrometer: Structural Analysis and Identification of Sialyl Linkage Isomers

Focusing on a blood serum-derived N-linked  glycan, this article introduces an example in which sialic acid residue was stabilized by using the sialic acid linkage specific alkylamidation (SALSA) method and detection and analysis were performed with a Shimadzu MALDImini-1 compact MALDI digital ion trap (MALDI-DIT) mass spectrometer. 

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Characterization of Glycan Binding Site of O-Linked Glycopeptides Using a MALDI-7090

Glycosylation Profile of IgGs Using a Linear Benchtop MALDI-TOFMS and Affinity Purification of Fc

Purification of enzymatically fragmented IgGs by affinity-beads enables batch analysis for the glycosylation using a bench-top MALDI-TOFMS, MALDI-8020. The MS resolution of  MALDI-8020 is sufficient to recognize three Fc that mainly differ in glycosylation. A statistical analysis by eMSTAT Solution enables a classification of three glycosylated Fc smoothly and  quickly, which could be applicable to QA/QC.

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Additional Applications

N-Terminal Amino Acid Sequencing of IgG Antibodies

N-Terminal Amino Acid Sequencing of IgG Antibodies

This article introduces an example of amino acid sequencing of mouse antibody IgG using the PPSQ-51A/53A Protein Sequencer isocratic system as an instance of N-terminal amino acid sequencing of biomedicines. 

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Peptide Mapping of Antibody Drugs by Nexera-i

Peptide Mapping of Antibody Drugs by Nexera-i

Peptide mapping by HPLC is one of the important quality assurance tests used for verifying the primary structure of antibody drugs. Typically, following enzymatic digestion of the antibodies, separation is conducted using a traditional reversed phase column. Due to the large number of peaks that require separation, the use of small-particle columns and core shell columns for peptide analysis has spread in recent years. In order to compare elution profiles for identity and mutation confirmation, a highly repeatable system is required.  Here, the Nexera-i is used in the analysis of IgG (human immunoglobulin G) tryptic digest.

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Analysis of mAb Aggregates by Nexera Bio UHPLC with a Shim-pack Bio Diol (Size Exclusion) Column

Analysis of mAb Aggregates by Nexera Bio UHPLC with a Shim-pack Bio Diol (Size Exclusion) Column

The production of mAb biosimilar is challenging, because various variations may occur in upstream and downstream processing(DSP). Protein aggregation is one of such variations ─ a biological phenomenon in which mAb accumulate and clump together. Aggregation is a critical quality attribute (CQAs), as the aggregates not only reduce the efficacy of mAb biosimilar drugs, but also can stimulate immunogenic responses, leading to various adverse events in treatment. 

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Optimizing Aggregate Analysis - Pore Size

Optimizing Aggregate Analysis - Pore Size

In SEC,smaller molecules enter the pores of the particle and proceed slowly along the axis direction of the column while molecules larger than the pore size are excluded from pores and elute at first from the column.In this application, we demonstrate the effect of pore size in SEC analysis. 

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Optimizing Aggregates Analysis - Mobile Phase

Optimizing Aggregate Analysis - Mobile Phase

Despite being a simple assay, mobile phase condition is often needed to be optimized to improve peak shape and resolution of proteins.In this study, we describe analysis of trastuzumab, an anti-HER2 mAb using Shim-pack Bio Diol column on Nexera Bio UHPLC.The effect of mobile phase composition (ionic strength and pH) on chromatographic separation of trastuzumab is presented in this application news.

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Charge Variant analysis of mAb Biosimilars by Nexera UHPLC with a Shim-pack Bio IEX Column

Charge Variant analysis of mAb Biosimilars by Nexera UHPLC with a Shim-pack Bio IEX Column

In this study, we describe both salt-gradient and pH-gradient methods for separating the charge variants of bevacizumab biosimilar using Shimadzu Nexera UHPLC and a Shim-pack Bio IEX column (4.6 mm x100 mm, 3 μm).

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Exploring Factors that Influence Charge Variant Analysis of mAbs with Shim-pack Bio IEX Columns using Salt Gradient Elution

Exploring Factors that Influence Charge Variant Analysis of mAbs with Shim-pack Bio IEX Columns using Salt Gradient Elution

In this study, we describe a charge variant analysis of Trastuzumab using salt gradient with Shimadzu strong cation-exchange column, Shim-pack Bio IEX SP-NP. The separation efficiency under different gradient slopes, gradient times and flow rates are discussed.

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