Modern biopharmaceutical drugs, such as monoclonal antibodies (mAbs) are highly complex molecules manufactured inside living cells. Characterizing and monitoring such compounds can be extremely challenging, but today’s use of sophisticated mass spectrometry offers a powerful solution.
Multiple Attribute Method (MAM) analysis (via peptide mapping) serves to provide significant amounts of information for biotherapeutic drugs, both reducing the number of analyses and different experiments required to be performed while increasing the product quality profile.
Pharmaceutical Processing recently reached out to a top expert on MAM, Jonathan L. Josephs, Ph.D. at Thermo Fisher Scientific, to provide keen insight into the techniques and technologies involved in using MAM. Josephs is the Director of Global Marketing and Strategy, Pharma and BioPharma, in the Life Sciences Business Unit of the Chromatography and Mass Spectrometry Division.
Q. Please explain the basics behind the Multiple Attribute Method and what it has meant for the further development in the manufacture of biopharmaceuticals.
Jonathan L. Josephs: The Multiple Attribute Method is explained in detail in the seminal paper by Richard Rogers et.al, published in 1995.
Biotherapeutics are large complex biological species that do not typically exist as a single homogenous chemical entity. They exist as a heterogenous mixture of closely related species. Some aspects of the heterogeneity such as the glycosylation pattern of monoclonal anitbodies (mAbs) is an inherent property of the biotherapeutic and may change due to changes in the expression system. Other changes such as post translational modifications of individual amino acids through, oxidation, deamidation, isomerization, etc. may take place during manufacture and storage. Still others, such as sequence variants, may take place during the transcription process.
These variations and modifications (attributes) may be most easily monitored at the peptide level after complete digestion of the biotherapeutic protein with a peptidase such as trypsin. The peptides are then analyzed by reverse phase chromatography coupled to High Resolution Accurate Mass (HRAM) mass spectrometry. The technique allows both the identification and relative quantitation of related peptides and therefore the determination and monitoring of attributes.
During the ‘Discovery and Development’ of a biotherapeutic the team of scientists determines which of these attributes affect safety and/or efficacy of the biotherapeutic. These are referred to as Critical Quality Attributes (CQAs) and are typically the attributes that would be monitored during process development, manufacturing, and ultimately in a Quality Control (QC) setting.
Q: Briefly describe the key steps available via MAM—monitoring, analysis, CMC (chemistry, manufacturing, and controls), verification, QC, other steps.
Josephs: The MAM workflow is divided into two main segments:
The first is a ‘Discovery’ experiment. The potential biotherapeutic protein is digested (typically with trypsin) and then analyzed by reverse phase LC/MS/MS using an Orbitrap mass spectrometer using data dependent acquisition. The MS1 scans from the experiment allow for component detection, preliminary identification, and relative quantitation. The MS2 scans provide high confidence in the peptide identifications. The data is processed with software in an unbiased fashion providing an exhaustive list of all attributes and their relative abundance across a wide concentration range.
Based on the knowledge of Discovery and Development scientists and the stage of development the list of attributes that are desired to be monitored is created and an MS1 method is built to monitor the relative quantitation of these attributes in a targeted fashion using extracted ion chromatograms with narrow extraction windows from the MS1 only dataset.
During clone selection and process optimization, a large number of attributes may be monitored and since the relative quantification is achieved from a full scan MS1 HRAM dataset, all the attributes are contained in the data and it is the building of the processing method that determines which are actually monitored. This enables development scientists to monitor as many attributes as they determine to be necessary. As the process moves downstream into manufacturing, it is typical to determine that only a smaller number of attributes need to be monitored. These Critical Quality Attributes (CQAs) are then built into a more focused processing method and based on process variation and the criticality of these attributes limit ranges may be set.
Moving into QC and release testing the method with defined limits is used to release batches of drug substance and may also be used for stability studies of drug substance and drug product. This final method is a fully compliant one with full audit trails processing and is fully automated such that samples are processed automatically and verified against pass/fail criteria without user intervention.
Q: When did Thermo Fisher Scientific get involved in MAM? What led to that original decision and what type of commitment was needed in technology, staff training, and product development?
Josephs: Thermo Fisher Scientific first became involved with MAM through a collaboration with Amgen that started back in 2012. Amgen investigated a number or mass spectrometry hardware platforms for this approach and determined that HRAM with at least 120K resolution (m/z 200 FWHM) was required to reliably quantitate attributes based on MS1 datasets.
The Orbitrap technology is inherently suited to relative quantitation based on HRAM MS1 datasets. Product development has been mainly on the software side to provide a streamlined fully CFR 21 part 11 compliant software with full audit and reporting functions for the QC lab.
Staff training involves making sure end users are familiar with the software for use in the QC environment. Typically, Discovery and Development scientists developing these methods for use in the QC labs have a higher degree of experience in mass spectrometry and are fully aware of the need to operate the mass spectrometer at high resolution settings (120K) and use narrow isolation windows to ensure selective and accurate quantitation.
In addition, they are familiar with how to use charge state and isotopic distribution to provide a further layer of confidence and security around the measurement. This is a feature that is inherently built into the software. Beyond selective, accurate, and confident relative quantitation of targeted analytes, a requirement of a release assay is to provide a measure of purity. A target analysis by its intent only measures what is targeted. To measure purity you must measure everything.
The acquisition of full scan HRAM datasets allows for ‘New Peak Detection’ comparison of a new sample with a reference sample employing differential analysis to detect any component within the sample that has changed by more than a specified fold change. The component detection algorithms employed by the software to conduct the analysis greatly benefit from instrumentation that produces data with very high resolution and mass accuracy.
Q: In the past, optical detection was employed, but in recent times organizations have moved toward incorporating mass spectrometry. What does mass spectrometry deliver that optical detection cannot?
Josephs: Optical detection has traditionally been employed in conjunction with various chromatographic techniques. They are essentially a bulk measurement of a protein. They do not reveal individual critical quality attributes with site specificity.
By way of example ‘Charge Variant Analysis’ by Ion Exchange Chromatography or Capillary Electrophoresis is very efficient at separating protein variants by charge but provides no indication of what modification has caused the difference. If two different Asparagine residues undergo deamination each would cause a single shift to an acidic variant but you would not know which amino acid in the sequence had been modified.
The MAM approach will tell you precisely how much of each site has undergone change. This is very important because the effect on safety/efficacy for each site could be very different.
Optical methods require that all components be fully chromatographically resolved in order to accurately measure them. The extra dimension of ‘separation’ afforded by mass spectrometry detection results in much more selective methods with much higher ‘peak capacity’ than chromatography alone. The higher the mass resolution and mass accuracy of the method, the greater the selectivity and ‘peak capacity.’
Q: With the trend toward more biosimilar development, what are the challenges that remain even after employing mass spectrometry for manufacturers in maintaining consistent and highly specific analytical data through the development process?
Josephs: With the acquisition of HRAM datasets for characterization and comparison of samples there is a greater burden on the data systems for processing and interpreting the data. Traditional Chromatography data systems are built around 2 dimensional data sets (Time-Intensity).
The introduction of a third dimension with mass spectrometry detection makes the datasets more complex and larger along with holding so much more information. The challenge then becomes how to best use these richer data archives.
Q: While certain native proteins are excluded from the final drug product through a series of purification and chromatographic polishing steps, some may still remain and adversely affect a drug’s safety. Does MAM help eliminate all such native proteins, or do some still make it through and, if so, how is that handled?
Josephs: MAM is an analytical assay and not a purification process. However, the power of the MAM driven by HRAM peptide mapping is that it can detect and measure almost any protein modality that is amenable to digestion—for example, when a biotherapeutic protein sample is digested by trypsin. In addition to all of the tryptic peptides corresponding to the biotherapeutic protein, representing all of the quality attributes there will be tryptic peptides that result from digestion of host cell proteins (HCPs).
This can be determined in a discovery experiment much like quality attributes are initially determined and then built into a list (or library) so that they may be subsequently monitored in a targeted method as a standard part of the MAM without any additional cost/complexity to the standard assay.
Q: In addition to proteins, does MAM also help in the identification and monitoring of the larger peptides?
Josephs: MAM is applicable to any protein or large peptide that can be digested as well as to smaller peptides by enzymes such as trypsin. It should be noted that Orbitrap instrumentation is very capable of providing isotopically resolved data for proteins up to 50 kDa.
Many larger peptides may be very well monitored without the need for digestion. Depending on the ‘larger peptide’ in question, the best analytical approach may be through MAM, an intact approach, or possibly a combination.
Q: The need for precise analytics sounds like it can become quite costly in a biopharmaceutical environment that requires ongoing testing and analysis. Has the new technique cut costs significantly for manufacturers?
Josephs: Unit costs for mass spectrometers are typically significantly higher than for optical detectors. Mass spectrometer costs have come down in actual dollars and there are lower performance mass spectrometers now being offered at the price point of an LC system. Higher performing Orbitrap-based HRAM instruments are still significantly more expensive than an optical detector. However, a single MAM based LC/MS system may replace multiple optical based assays/systems and so the total cost of monitoring attributes by MAM rather than by the traditional assays that it improves upon can lead to direct cost savings.
True cost savings of course go well beyond the direct analytical costs: the financial advantages of bringing an innovator or biosimilar drug to market sooner; the ability to improve manufacturing processes to generate higher yields and tighter specifications. To release batches faster and with higher confidence not only result in fiscal benefits, but they provide a higher degree of patient safety.
Can smaller-size CMOs and others also benefit from MAM?
Josephs: The MAM approach provides greater insights, speeds up process development and manufacturing, and raises the quality of understanding of released batches. It is, therefore, of broad applicability across organizations of all sizes.
Q: Molecule characteristics can vary at multiple production sites even within the same company for the same drug. How is the MAM data compiled and shared when multiple sites are involved in biopharmaceutical development and complex molecular characteristics?
Josephs: MAM data acquisition and processing uses established CFR 21 part 11 enterprise based software that can be deployed across organizations.
While data is acquired into a ‘vault’ to ensure raw data integrity, raw and processed data may be shared throughout or between organizations.
Q: How has MAM aided companies in the preparation of data for submission to regulatory authorities? When different regulatory bodies are involved (FDA, EMA, etc.) does that complicate the data compilation methods so each data set satisfies the different regulatory authorities?
Josephs: For regulatory filings there have been attempts by regulatory agencies to harmonize certain aspects. The requirements of each agency do differ and any one filing will be adjudicated by potentially different reviewers. The general guidance often provided by agencies is on a ‘case-by-case basis’ and ‘based on scientific merits.’
In general terms, MAM is no different than any other analytical assay used in a filing. If it meets the requirements for the attributes being measured, and measurement of those attributes answer reviewers concerns for safety and efficacy, it is being seen as a scientifically sound approach.
Essentially, MAM is a ‘platform method’ and therefore once established should not be any more complicated to use in a filing than any other accepted assay.
Q: On a quantitative basis, how has MAM improved the instances of FDA, EMA, and other regulatory approvals since the development of the technique?
Josephs: This question really is not easy to answer. Over time the agencies have been setting higher standards for submissions than were previously acceptable.
Therefore, it is really not possible to answer this question.
The agencies are asking for more information and the MAM method can be more informative. It, therefore, is well aligned with the agencies’ desire to use state-of-the-art analytical methods.
Q: How closely do the regulatory authorities monitor the MAM technique in use? Do they inspect it regularly or require certain procedures to validate the technique?
Josephs: The regulatory agencies do not set different criteria for how closely they monitor any particular analytical technique.
They ask scientific and technical questions and they also conduct site inspections for compliance regardless of what analytical technique might be employed.
Q: Mass spectrometry’s use has expanded in biologics development. Are there other areas on the radar screen for this technique in pharma in the future?
Josephs: Mass spectrometry has in general been showing ever increasing applications in Pharma and BioPharma.
MAM is applicable across the Discovery to Development continuum through to manufacturing.
Q: Beyond biopharmaceuticals, is MAM being targeted toward other markets going forward?
Josephs: MAM is essentially a modality independent analytical technique applicable to all proteins and, as such, could be applied wherever detailed analysis of proteins is required.
This story can also be found in the March 2018 issue of Pharmaceutical Processing.