A Comprehensive CDMO Perspective from Richter BioLogics
By Dr. Maja Erdmann, Daniel Goetz, Dr. Daniela Stummer and Dr. Ingo Goldbeck at Richter BioLogics
In biopharmaceutical production, a thorough understanding of analytical processes and target molecules is essential for ensuring patient safety, as well as maintaining consistent and reliable product quality. Mass spectrometry (MS) represents a powerful toolbox to assess this knowledge and partially even compensates the need for a bundle of methods covering different analytical parameters due to its highly versatile applications. While mass spectrometry is often applied in the developmental phase, its use in biopharmaceutical routine testing is often hampered since requirements for specialized knowledge and GMP compliance are significant hurdles.
At Richter BioLogics, a leader in the microbial Contract Development and Manufacturing Organisation (CDMO) space, these hurdles were taken, opening the way to support development, production and release of biopharmaceutical products at multiple levels with an ESI-TOF (Electrospray Ionisation – Time Of Flight) based LC-MS (Liquid Chromatography-MS) system as a single device, beginning with characterization of upstream process components up to GMP compliant release testing of large biomolecules.
The implemented ESI-TOF LC-MS system thus covers a wide range of applications over the while product lifecycle, providing additional benefits to complete the analytical portfolio.
“Knowing your molecule” is a pivotal requirement in biopharmaceutical industries to ensure high product quality and patients’ safety. Understanding the properties and critical quality attributes (CQAs) of target molecules and their behaviour throughout the production processes allows decision about and setup of a suitable analytical panel for product characterisation, which provides the basis to deliver complex biomolecules with highest quality and sufficient yields by the most efficient processes in biopharmaceutical production. The need to characterise production processes and target molecules is defined in ICH Q14,1 stating that process and product understanding should be the first step in the analytical lifecycle. In biopharmaceutical production, molecules and processes are therefore characterised by a broad analytical portfolio from development to final release testing, covering analytical parameters such as content, potency, identity, purity, process and product-related impurities, which are generally addressed using various analytical equipment and techniques.
The analytical panel may vary throughout a product’s life cycle, depending on the complexity and aim of sample characterisation. Especially in the development phase, LC-MS (liquid chromatography mass spectrometry) is often applied for purity evaluation and characterisation, since it allows direct insights into the molecular composition of formulated bulk, in-process samples and its impurity profiles, and thus represents a suitable tool to gain product and process knowledge in a timely and efficient manner.
One of the most striking benefits during this phase is the identification of the product as well as product-related variants and modifications on the molecular level at various process steps and under different process settings. Thus, LC-MS represents a valuable tool, not only to optimise processes, but also to gain knowledge on product quality characteristics and main impurities as well as degradation pathways, which can support the definition of critical quality attributes (CQAs) and the set-up of an analytical panel. Furthermore, LC-MS provides valuable information upon implementation and development of a wide panel of LC methods, opening the opportunity to identify peaks and to verify peak purity. The obtained qualitative data are beneficial to understand the product on a molecular level, identify CQAs and gain insight into process understanding.
Furthermore, process understanding can be improved using the LC-MS system as a semi-quantitative tool on a small molecule level at the very beginning of process optimisation, the upstream processing (USP), to improve product quality and yield.
Besides these scientific advantages, LC-MS analyses can be performed within short time frames and even compensate the need for a wide analytical panel for process and product characterisation. Due to this, LC-MS provides significant benefits for the improvement of time and costs during development and production.
Despite these advantages applied for development purposes, the impact of LC-MS-based characterisation in a GMP-related context is often limited due to a constrained compliance of equipment, software and methods. Moreover, most mass spectrometry devices require a specialised team with relevant expert knowledge to develop and validate the required LC-MS methods. Nevertheless, the capabilities that LC-MS offers in this field are no less valuable than in areas of development and can provide a versatile tool in fields of method validation, release testing, reference standard characterisation, as well as stability studies, as soon as the GMP-relevant aspects are fulfilled. In this context, the recently updated ICH harmonised guideline Q2(R2) for validation of analytical procedures sets the validation design for quantitative LC-MS methods, further promoting LC-MS in GMP applications and reducing efforts and, thereby, costs for GMP-compliant implementation.
The following chapters provide exemplary insights into the individual applications of LC-MS analyses as well as the GMP readiness process.
Application of lC-MS in Upstream Bioprocess Development (Media Screening)
The upstream process (USP) at the very beginning of biotherapeutic production already sets the course for successful mid- and downstream processing, impacting yield, as well as quality of the final biopharmaceutical product. Thus, optimisation of the fermentation during USP, including, for example, feed media composition and feeding strategies for improved cell health and growth, as well as product titre and quality, has an essential impact on the final product.
At Richter BioLogics, the acquired LC-MS system has been established for quantitative monitoring of key nutrients and metabolites (e.g. amino acids and vitamins) in cell culture media and supernatants following the LC-MS system manufacturer’s application notes and amino acid/cell culture standard. While common USP optimisation strategies often rely on general aspects, such as cell growth curves, gas and carbon source profiles and product titre, the additional monitoring of specific components of cell culture supernatants, which remain uncertain in common approaches, can significantly speed up USP optimisation by elucidating critical key components.
Cell culture components are identified by their measured retention time and mass on the high-resolution ESI-T0F mass spectrometer in full scan mode and small molecule acquisition range (50-800 m/z). This allows, for e><ample, for the quantification of isobaric components, such as Leucine and lsoleucine (see Figure 1, a and b). Cell culture supernatants are diluted appropriately and the calibration curve is set up based on the manufacturer’s instructions. Independent of the calibration curve unit as amount or concentration (see Figure 1, c), the LC-MS software provides the relevant output (e.g. molar concentration of a component in the undiluted sample). Quantification of key components over time and by direct comparison between fermentation runs delivers valuable knowledge for USP optimisation, which is also graphically supported by the LC-MS software (see Figure 1, d).
As illustrated in Figure 1, LC-MS is a method which can selectively identify and quantify target cell media components in complex cell culture supernatants, revealing differences in their levels of consumption throughout fermentation processes.
Accordingly, LC-MS allows for a more target-oriented and streamlined development of early biotherapeutic processes, consequently leading to improved yield and quality of the final product in a time- and cost-efficient manner.
Figure 1: Media Screening examples for Isoleucine quantification in two comparative fermentations (USP1 and USP2) performed with different cell culture media. a, Zoom of an extracted ion chromatogram (XIC) of isobaric amino acids Ile and Leu; b, Mass spectrum of Ile (protonated form in positive polarity and isotope peaks highlighted in green); c, Calibration curve of Ile; d, Trending plot bar chart of Ile responses for selected injections (calibration curve, samples of USP 1 and 2).
Application of LC-MS in Downstream Bioprocess Development
The benefits of LC-MS analyses to give direct insights into molecular details of samples and provide additional process knowledge were successfully applied at Richter BioLogics by identifying unknown peaks observed during downstream process monitoring. The respective species was observed in analytical UV-based RP-UPLC of in-process samples during the optimisation of several process parameters. Since the nature of this species and its impact on process-accompanying content determination by UV-based RP-UPLC was unknown, further characterisation was required. To this end, the UV-based RP-UPLC method was transferred to the ESI-TOF LC-MS system and samples of interest were analysed in intact mass modus (high range covering 400-7000 m/z). Deconvolution by the MaxEnt1 algorithm of mass spectra, averaged at peak maxima, revealed average molar masses of relevant species observed in analytical RP-UPLC. Based on these data, the relevant species was identified as a truncated isoform and its impact on content determination was assessed accordingly. The identification process of unknown peaks in RP-UPLC is illustrated in Figure 2.
LC-MS analysis provided the basis for a time- and cost-efficient manner by targeted and scientifically based development. Thus, this case illustrates the power and advantages of LC-MS at early stages of process development, which are equally suitable and transferable to other questions in process development.
Besides its application for identification purposes, LC-MS can also be applied as a valuable tool to sensitively quantify process related impurities. An LC-MS workflow for quantitation of residual albumin was developed at Richter BioLogics on the ESI-TOF LC-MS based on intact mass modus (high range covering 400-7000 m/z), covering a linear range of 2-50 ng albumin. Optimisation of the method involved the gradient, quality of reagents and consumables, detector settings and evaluation procedures based on total, as well as extracted ion chromatograms (TIC, XIC). The accuracy of the method was verified using different control samples.
The implemented method opens the possibility to cover residual albumin quantification during development, but also in release testing of respective APIs in a fast, sensitive and specific manner.
Figure 2: Exemplary illustration of LC-MS based characterisation process. a, Zoom of UV-based chromatogram obtained by RP-UPLC; b, 3D Mass spectrum components observed in RP-UPLC; c, Averaged raw mass spectrum og main peak observed in RP-UPLC; d, Deconvoluted mass spectrum af main peak observed in RP-UPLC.
Requirements for the Operation of Mass Spectrometry in a GMP Environment
The power of LC-MS for analytical support during the developmental phase is based on its inherent analytical advantages to directly assess knowledge about product characteristics and product-related impurities, making it a versatile tool to report purity as well as identity data for a wide panel of samples and studies. These advantages are, in principle, equally applicable for product evaluation in a GMP-related context. Nonetheless, despite the versatile scientifically sound application options, usage of LC-MS methods in a GMP environment can be limited since application of instruments and techniques in a regulated environment requires defined prerequisites regarding equipment, software and methods.2,3 These prerequisites include aspects of instrument and software qualification, data integrity concepts, as well as training of operators and reflect a complex setup especially for LC-MS systems.
Analytical systems with GMP-relevant applications must fulfil several requirements. First of all, documented evidence must be in place verifying the suitability of the system for its intended purpose with regard to instrumental settings, IT infrastructure, data integrity and related documentation. These requirements are documented and verified during the qualification process, including phases of user requirements definition (URS), design (DQ), operational (OQ) and performance qualification (PQ).
Furthermore, a detailed description of the system, the workflows, maintenance and qualification, and a review of analytical and metadata must be in place and QA (quality assurance) approved prior to GMP usage.
As the first step in extending Richter BioLogic’s analytical portfolio using mass spectrometry, we defined and documented the user requirements (URS) for the planned LC-MS system according to our internal SOPs. The following aspects were covered:
- Technical prerequisites
- Requirements regarding instrumental settings and ranges for all LC-MS modules
- Requirements related to the infrastructure
- Provider of the instrument
- Provision of documentation
- Quality system and quality management of the provider
- IT aspects
- Electronic records and signatures
- User profiles
- Data storage
Based on the above-mentioned URS documentation and prerequisites, an LC-MS ESI-TOF equipped with a software generally compliant with 21 CFR part 11 was chosen as a candidate and evaluated regarding compliance with the requirements in the process of design qualification. All aspects of the URS were subsequently verified during operational and performance qualification either by the external provider or by internal assays.
Additionally, a working instruction was issued covering and describing the following aspects in a detailed manner:
- Description of the LC-MS system and its IT environment
- Description of analytical procedures
- General chapter
- Workflows describing intact mass as well as peptide (mass) mapping analysis
- Requirements for data reporting
- Requirements for data review, including data integrity aspects
- Administrative aspects
- Definition of policies
- Folder structure
- Audit trail settings
- User profiles and user privileges
- Data storage and back-up
- Aspects of qualification and maintenance
Based on the above-mentioned documentation, GMP compliance of the LC-MS system with regard to instrumental settings and ranges, as well as performance and review of analyses, data integrity and administration is ensured.
Application of LC-MS in GMP-relevant Aspects
Successful verification and documentation of instrumental and software parameters, as well as available and clearly defined procedures for application of an LC-MS system, paves the way for a broad panel of LC-MS applications in GMP-related topics.
For example, the benefits of mass analyses are essential for the characterisation and release of reference standard material, since primary reference standards used in the QC environment of API (active pharmaceutical ingredient) production are recognised as presenting the highest metrological qualities with their property values accepted without reference to other standards (see Ph. Eur.5.124). For their qualification (as well as batch comparison in the case of standard replacement), they are extensively characterised (see ICH Q72), which generally includes additional analyses such as determination of molecular weight, primary sequence, N-/C-termini, (post-translational) modifications and higher order structure (e.g. tertiary structure by disulphide bridging). As these parameters can be addressed by intact mass and peptide (mass) mapping workflows, LC-MS essentially contributes to primary reference standard qualification.
Moreover, OOX events such as out-of-specification, expectation or trend occasions have to be clearly documented and thoroughly investigated in a GMP environment.5, 6 LC-MS can provide valuable information for the investigation process, complementing the insights gained by the concerned method and contributing to the final conclusion of the OOX procedure.
Furthermore, intact mass as well as peptide (mass) mapping analyses represent powerful techniques to identify and report CQAs for batch release analyses of biopharmaceutical products, covering purity and identity quality attributes, providing direct insights into product quality on a molecular level. Especially, simultaneous detection and quantitation of product-related impurities in formulated bulk samples enables LC-MS analyses to be a suitable tool for monitoring sample purity over time, providing a solid data set for evaluation of stability studies.
Summary
Mass spectrometry presents a high-resolution, sensitive and extremely flexible and diverse technique, gaining increasing influence in the biopharmaceutical industry, particularly within the CDMO market, where Richter BioLogics stands out as one of the leaders. Although its application in a GMP environment requires comprehensive activities such as system qualification and compliance in data integrity, the advantages of utilising this technique, once established under GMP, are striking; process development, production and release of biopharmaceutical products guided at multiple levels throughout the entire process and product life cycle; covering small to large biomolecules and various analytical parameters in a time-and cost-efficient manner. Beginning with upstream process optimisation up to GMP-compliant release testing of large biomolecules, mass spectrometry efficiently increases product quality and thus, patient safety.
References:
- Visit: International Council for Harmonisation (ICH): ICH Q14-Analytical procedure development, 12.11.2023, accessed 07.08.2025 from www.ema.europa.eu/en/documents/scientific-guideline/ich-q14-guideline-analytical-procedure-development-step-5_en.pdf
- Visit: International Council for Harmonisation (ICH): ICH Q7 – Good Manufacturing Practice for Active Pharmaceutical Ingredient, accessed 07.08.2025 from www.ema.europa.eu/en/documents/ scientific-guideline/ich-q-7-good-manufacturing-practice-active pharmaceutical-ingredients-step-5_en.pdf
- Visit: USP General Chapter <1058> Analytical Instrument Qualification – Compliance Compendium; 2017.
gc-1220-pre-post-20210924.pdf - Visit: European Pharmacopoeia, Chapter 5.12. Reference Standards. Ph. Eur. Suppl. 11.8. Strasbourg, France: Council of Europe; 2025.
- Visit: EudraLex-Volume 4: EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use. Part I: Basic Requirements for Medicinal Products. Chapter 6: Quality Control. Brussels: European Commission; Revision March 2014, accessed 07.08.2025 from https://health.ec.europa.eu/document/download/ c74c8720- 27bf-4252-808f-d65a 206a90bb_en
- Visit: Laboratory Management Guidance: Out of Expectation (OOE) and Out of Trend (OOT) results, ECA Analytical Quality Control Working group; 2015.
Dr. Maja Erdmann is a biochemist and senior scientist in the LC department of Richter BioLogics, specialized in LC-MS analyses of biopharmaceutical products.
Daniel Goetz is a pharmaceutical biotechnologist and scientist in the LP department of Richter BioLogics, focusing an LC-MS analyses of biopharmaceutical substances.
Dr. Daniela Stummer is a chemist and senior scientist in Department “Separation Techniques” at Richter BioLogics, specialized in UV- and MS-based LC analyses of biopharmaceutical products.
Dr. Ingo Goldbeck is a biochemist and Head of Department “Separation Techniques” at Richter BioLogics GmbH.
Quelle: European Biopharmaceutical Review | Summer 2025
