Biopharmaceutical Multi-Attribute Method

The ultimate goal of biopharmaceutical development and manufacturing is to deliver the highest quality product, at the lowest cost, in the shortest time possible. However, large molecule drugs are highly complex, leading to analytical challenges throughout the development pipeline to quality control and release. Monoclonal antibodies (mAbs) and other complex biological drug products, such as antibody-drug conjugates (ADCs), must be exhaustively characterized to ensure the safety and efficacy of a batch before release. The Multi-Attribute Method (MAM) aims to address these challenges using the power of high-resolution accurate mass mass spectrometry to improve process control for biopharmaceutical manufacturing.

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What is the Multi-Attribute Method?

The Multi-Attribute Method (MAM) is a peptide mapping based method which takes advantage of high resolution mass spectrometric data. High-resolution mass spectrometry provides the accurate mass information aiding identification of product quality attributes and consequent quantitation and monitoring, which leads to a more comprehensive biomolecule process understanding, allowing for better process control for manufacturing. It is a relatively new analytical approach that is particularly well suited to biotherapeutics QC (publication in mAbs [2015]).

What is the Multi-Attribute Method?

MAM is based upon traditional peptide mapping. To begin building the method, the biotherapeutic must first be digested into peptides. This critical step requires 100 percent sequence coverage, high levels of reproducibility, and minimal process-induced modifications (for example, deamidation).

The resulting peptides are separated using liquid chromatography (LC) and detected using high resolution accurate mass (HRAM) MS. The data is then processed using sophisticated software tools.

HRAM MS for MAM provides a comprehensive view of the critical quality attributes (CQAs) present in biotherapeutics, down to the individual amino acid sequence of each molecule. Detailed information can be obtained on post-translational modifications (PTMs), glycoprotein structures, the presence of low-level sequence variants, and minute concentrations of process impurities. High-resolution accurate mass data makes access to this detailed information possible without the need for full chromatographic separation.

Another key component of MAM is new peak detection, which enables new peaks (impurities) to be detected when compared to a reference sample. This data analysis step is essential for using MAM as a QC release method. MAM is a singular approach that has the potential to consolidate multiple analyses from QC to batch release, enabling scientists to work towards consistent biotherapeutic products across the process, in line with Quality by Design (QbD) principles.

A well-developed MAM enables identification, quantitation, and monitoring of multiple CQAs simultaneously as well as new peak detection. The benefits of an optimized and standardized Multi-Attribute Method for characterization of biologics and later stage quality control include:

  • High-resolution MS data for absolute identification and confidence in analytical control over quality
  • A single method providing the same or superior product quality information instead of many traditional methods and techniques
  • A single assay that offers cost and time savings, as well as fewer SOPs and instruments to maintain
  • Offering consistently reliable analytical processes on multiple instruments across different development sites, from research and development to manufacturing and QC
  • Accepting and standardizing MAM from development into QC space will offer peace of mind when preparing regulatory filings

The Thermo Scientific HR Multi Attribute Method includes all the abovementioned benefits in a locked down workflow product.

Thermo Scientific HR Multi Attribute Method

Benefits of a Multi-Attribute Method Workflow

A MAM workflow provides comprehensive characterization of therapeutic drugs in line with the QbD principles developed by the U.S. FDA and EMA. It also helps reduce the number of required assays and analytical complexity by using high-resolution mass spectrometry.

Comprehensive characterization in line with Quality by Design principles

Regulatory agencies such as the U.S. Food and Drug Administration (U.S. FDA) and the European Medicines Agency (EMA) have adopted principles to enhance discovery, development, and manufacturing of therapeutic drugs, referred to as QbD.

The QbD approach aims to ensure quality of therapeutics using analytical, statistical, and risk management methodologies in their design, development, and manufacturing. QbD guidelines require a comprehensive understanding and thorough characterization of a drug’s quality attributes at the molecular level, and, with further studies, allows scientists to determine CQAs needing to be monitored assuring therapeutic product quality, safety, and efficacy throughout development and manufacturing lot release. A comprehensive, site-specific characterization is performed during the early phases of development. The information from that characterization is built into quality monitoring methods, allowing streamlined product and process development practices, building in quality as early as possible.

Fewer assays and reduced analytical complexity with high resolution MS

Characterization and quality control of biotherapeutics typically requires multiple labor-intensive or time-consuming analytical techniques; for example, cation-exchange chromatography, imaging capillary isoelectric focusing, and capillary electrophoresis sodium dodecyl sulphate. Unfortunately, each technique typically provides information about only one, or possibly a handful, of CQAs – and only after significant analysis. Traditionally, multiple assays are used in combination to identify and monitor CQAs individually. Many are profile-based and are not capable of identifying and quantifying at the molecular attribute level.

HRAM-MS coupled with high performance separation represents the cutting edge of biotherapeutic characterization, not only because it offers high-resolution data and impressive levels of sensitivity, but also because it increases confidence in results. HRAM MS has been more frequently adopted and used routinely in discovery analytical labs, as it is seen as a more complex technique which requires skilled operators. These skilled users operate the instruments and perform necessary, but often complex, data processing workflows. However, due to the complexity of today’s biopharmaceuticals, high-resolution data is required to accurately and confidently answer questions about quality attributes. The barriers to adopting MS beyond discovery and implementing it for QC and lot release are increasingly debated, but its power to support quantitative peptide monitoring in a reliable fashion is clear. New and improved analytical systems and software can be leveraged to support biopharma companies moving in this direction.

How can HRAM MS reduce the number of individual CQA assessments?

Using an appropriately developed MAM, the number of individual tests (e.g., CEX, CE-SDS, HILIC, ELISA) for specific CQAs can be decreased, as shown in the table below.

Adapted from Rogers, R. et al. MS in QC: A Single Multi-attribute Method for Quality Control and Release Testing of Biologics. Presented at CASSS MS 2013, Boston, September 24 2013.

 Yes = YesNo = NoSometimes = Sometimes

To adequately cover the numerous different critical quality attributes required during QC analysis, several different analytical strategies are typically performed. Many of these analyses are time intensive and only serve to provide one piece of information.

Multi-attribute method (MAM) analysis (via peptide mapping) serves to provide a significant amount of information for biotherapeutic drugs, both reducing the number of analyses and different experiments required to be performed while increasing the product quality profile.

Implementing a Multi-Attribute Method workflow

A well-developed MAM workflow enables characterization of biologics with ease and allows potential critical quality attributes (CQAs) to be monitored throughout every stage of the drug development process, while also allowing for purity testing using new peak detection (NPD).

All key steps of the MAM workflow need to be optimized to achieve the most reliable results. These results are crucial for informed decision-making during biopharmaceutical development and for regulatory filings.

Implementing a MAM workflow

The key components of a MAM workflow are:

  • Sample preparation – enzymatic digestion of a protein into distinct peptides
  • Peptide separation – liquid chromatography separation
  • High-resolution accurate mass (HRAM) mass spectrometric detection of peptides
  • Instrument control, data recording, and data analysis (automated sample analysis, peptide identification and quantitation)
  • Checking system/analytical performance and generated data reliability with system suitability
  • Service and support

MAM is based on the principles of peptide mapping, which takes advantage of the accurate mass measurement of unique protein fragments produced by highly specific enzymatic digestion. Consequently, for a successful MAM implementation, the therapeutic protein must first be digested into its constituent peptides.

Fast and reproducible standardized protein digestion is key for a well optimized MAM. Digestion is most often performed with specific proteases like trypsin, chymotrypsin or, in some recent publications, pepsin. It can be performed according to an in-solution digestion protocol or with a sample preparation kit, which can be easily automated with minimal user intervention and high reproducibility.

Trypsin is the most commonly used enzyme for digestion because it produces peptides in the size range (~4–45 amino acid residues) that is most efficient for protein identification (400 <m/z <5000). At this size range, the generated peptides exhibit sufficient specificity, but are still a manageable size for mass spectrometric analysis.

Thermo Scientific SMART Digest Kits represent a significant advance in sample preparation for biopharmaceutical characterization. These kits use immobilized trypsin for fast and simple protein digestion with high reproducibility, high sensitivity, and superior data quality in a format compatible with automation.

A high-pressure liquid chromatography (HPLC) system is required in order to separate peptides at the required resolution and enable analysts to accurately identify and quantitate peptides. Today’s high-end ultra-high-pressure liquid chromatography (UHPLC) systems offer exceptional robustness, high gradient precision, improved reproducibility, and peak efficiency for the high-resolution reversed-phase peptide separations required for targeted peptide quantitation, a prerequisite for a MAM workflow. Thermo Scientific Vanquish UHPLC systems offer unprecedented robustness and reproducibility to fulfill these requirements and help implement a successful MAM workflow.

An important aspect of using a UHPLC system for the MAM workflow is choosing the correct UHPLC column, then installing, testing, and optimizing it for the workflow. Using the extended pressure capabilities of the UHPLC system enables it to operate at its full potential, including the ability use of a variety of UHPLC columns. Some columns contain 1.5 µm solid core particles, deliver exceptionally sharp peaks, maximal peak capacities, and remarkably low retention time variations. These features and capabilities enable reproducible peptide mapping for reliable batch-to-batch analysis during routine QC applications, leading to more accurate quantitation.

Thermo Scientific Acclaim VANQUISH C18 UHPLC columns, Thermo Scientific Accucore columns, and Thermo Scientific Hypersil GOLD columns can be used for peptide mapping of biotherapeutic proteins, as they offer excellent separations and help ensure sharp peaks are achieved to will help accurate quantitation. These columns offer steady retention times, so scientists can be confident in the identity of every peptide in discovery, development, and QC environments.

For more information visit Peptide Separation Solutions

The chromatographically separated peptides are analyzed in an HRAM mass spectrometer for identification. The successful identification of peptides depends on the performance of the mass spectrometer and its achievable mass resolution and mass accuracy. The more accurate the measured masses are the easier it is to determine which peptide and fragments they are coming from. With the help of specialized software algorithms, peptides and their modified counterparts can be identified. Peptide identification allows the protein’s primary structure to be mapped and confirmed and post translational modifications identified. In the next step, these modifications can be accurately quantitated, and risk assessed based on their therapeutic or harmful properties. Some modification may be chosen for further monitoring, allowing them to be controlled as product quality attributes.

As a result of its increased sensitivity, specificity and its ability to accurately identify and quantify peptides, HRAM MS has become the gold standard in peptide analysis for biotherapeutics. Low-resolution MS techniques offer lower specificity and cannot confidently determine some critical modifications, especially when there is no chromatographic separation between the modified and unmodified peptides.

For discovery peptide mapping experiments when building a MAM, peptides are analyzed through MS fragmentation steps in data-dependent acquisition mode, and accurate mass information followed by collection of peptide fragment information. This process enables confident peptide identification. For monitoring and quantitating these peptides in subsequent analyses, the information from the chromatographic separation, combined with the accurate mass information, provides sufficient and reliable confirmation of peptide identification. In this case, the quantitative experiments no longer require fragmentation data collection. They use the HRAM MS1-only trace for quantitation.

BioPharma Finder software includes capabilities to analyze data sets including full MS and MSMS fragment ion spectra, supporting the generation of a comprehensive peptide map. Ideally, this map represents 100% sequence coverage, matching up all significant attributes of the drug molecule. This information, together with any previous knowledge of the product regarding CQAs, are saved in a list for future routine analysis. The list must be processed using software capable of routine quantitation.

For compliance-ready QC monitoring, the data processing software, such as Chromeleon CDS, can:

  • operate the LC-MS system in a 21 CFR Part 11 compliant fashion
  • acquire high resolution MS1 only data on a peptide digest of all future lots of the biologic drug, automatically compare the relative abundances of CQAs between a reference standard (e.g., a chosen reference lot) and any new batches or lots (e.g., from testing a new manufacturing process)
  • detect any unexpected or unknown impurities
  • generate a customizable report complete with electronic signatures

The data system provides rich tools for LC-MS data analysis and a composite scoring algorithm to give a clear PASS or FAIL to the target attributes for the molecule, essentially democratizing peptide quantitation to a simple red/green report.

The software must support:

  • Discovery peptide mapping for identification of product quality attributes and allowing the user to build a peptide list for monitoring
  • Confirmation of amino acid sequence
  • Identifying the site and type of expected and unknown PTMs and providing the relative amount of the modification in the sample
  • Disulfide bond mapping
  • Determination of low-level impurities – sequence variants
  • Detection of sequence alterations – in stress samples, level of deamidation or oxidation

Instrument control and routine data processing

  • Data measurement control in a compliance-ready environment
  • Quantitation of peptides
  • Detecting new peaks
  • Reporting

A well-developed MAM process requires verification that it identifies attribute peptides correctly and quantifies them accurately. The system used for the MAM analysis, therefore, needs to be tested using an appropriate system suitability test and a corresponding test material. A commercially available peptide mixture may serve this purpose. It is preferable that the sample is not just a mixture of synthetic peptides, but a peptide mixture resulting from the digestion of an appropriate protein. The Pierce BSA Protein Digest Standard is often used by scientists in the BioPharma industry to verify the suitability of the LC-MS system for peptide mapping and quantitation.

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HR Multi-Attribute Method animation

Learn how innovation and monitoring strategies can reduce the number of tests and enhance the methodology of validating impurity.

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