Biopharmaceutical Multi-Attribute Method

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.

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.

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.

Critical Quality Attribute Assessment	Covered by MAM	SEC	CEX	rCE-SDS	nrCE-SDS	HILIC	ID ELISA	HCP ELISA
Aggregation			Indirect
CDR Conformers			Indirect
CDR Tryptophan Degradation		Indirect
C-terminal Amidation			Indirect
C-terminal Lysine
Cysteine Adducts
Deamidation			Indirect
Dimer
Disulfide Isoforms			Indirect
Disulfide Reduction
DNA
Fragmentation (Peptide Bond)
Fucosylation
Galactosylation
Glycation
HCP
High Mannose
Hydroxylysine
Identity
Methionine Oxidation
Mutations & Misincorporations
Non-consensus Glycosylation
Non-glycosylated Heavy Chain
N-terminal pyroGlutamate			Indirect
O-linked Glycans
Residual Protein A
Signal Peptide
Thioether
Trisulfide
Unusual Glycosylation			Indirect

= Yes

= No

= 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.

• Learn more about the development of a quantitative mass spectrometry multi-attribute method for characterization, quality control testing and disposition of biologics in this scientific article from Rich Rogers. (publication in mAbs [2015])

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.

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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.

• Application note: Increased Long-term Stability of Peptide Mapping using the Vanquish UHPLC System

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.

• Article: Mass spec weighs in on protein therapeutics

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.