Dynamic mass spectrometry probe for electrospray ionization mass spectrometry monitoring of bioreactors for therapeutic cell manufacturing

Chilmonczyk, Mason A.; Kottke, Peter A.; Stevens, Hazel Y.; Guldberg, Robert E.; Fedorov, Andrei G.

doi:10.1002/bit.26832

Cited by 11 publications

(18 citation statements)

References 47 publications

(98 reference statements)

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“…Furthermore, HCPs with functions in spindle segregation (sperm‐associated antigen 5) and other or unknown functions (filamin‐C isoform X1, 1,2‐dihydroxy‐3‐keto‐5‐methylthiopentene dioxygenase, UPF0160 protein MYG1) were expressed solely under Condition 2. Some of these HCPs may be indicator HCPs for a suboptimal USP and may allow quick process interference when monitored on site during fermentation for example, by MS‐monitoring of the bioreactor in the future. On the other hand, enriched functions of 46 HCP detected uniquely under condition 1 based on DAVID functional annotation can be linked to protein transport (e.g., coatomer subunits), cellular structure (tubulin, microtubule‐associated protein 1S) or protein transcription (e.g., 60S ribosomal protein).…”

Section: Resultsmentioning

confidence: 99%

Mass spectrometric evaluation of upstream and downstream process influences on host cell protein patterns in biopharmaceutical products

Falkenberg

Waldera-Lupa

Vanderlaan³

et al. 2019

Biotechnology Progress

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For production of different monoclonal antibodies (mAbs), biopharmaceutical companies often use related upstream and downstream manufacturing processes. Such platforms are typically characterized regarding influence of upstream and downstream process (DSP) parameters on critical quality attributes (CQAs). CQAs must be monitored strictly by an adequate control strategy. One such process-related CQA is the content of host cell protein (HCP) which is typically analyzed by immunoassay methods (e.g., HCP-ELISA). The capacity of the immunoassay to detect a broad range of HCPs, relevant for the individual mAb-production process should be proven by orthogonal proteomic methods such as 2D gel electrophoresis or mass spectrometry (MS). In particular MS has become a valuable tool to identify and quantify HCP in complex mixtures. We evaluate up-and DSP parameters of four different biopharmaceutical products, two different process variants, and one mock fermentation on the HCP pattern by shotgun MS analysis and ELISA. We obtained a similar HCP pattern in different cell culture fluid harvests compared to the starting material from the downstream process. During the downstream purification process of the mAbs, the HCP level and the number of HCP species significantly decreased, accompanied by an increase in diversity of the residual HCP pattern. Based on this knowledge, we suggest a control strategy that combines multi product ELISA for in-process control and release analytics, and MS testing for orthogonal HCP characterization, to attain knowledge on the HCP level, clusters and species. This combination supports a control strategy for HCPs addressing safety and efficacy of biopharmaceutical products. K E Y W O R D S biopharmaceuticals, ELISA, host cell protein, mass spectrometry, proteomics 1 | INTRODUCTION Recombinant monoclonal antibodies (mAbs) produced in Chinese Hamster Ovarian cells are among the most important biopharmaceutical drugs. Different sub-populations of the Chinese hamster ovary (CHO) cell line are commonly used in biopharmaceutical processes for mAbs 1 . mAbs are steadily secreted into the cell culture fluid (CCF) during the fermentation process. During different steps of the DSP mAbs are purified and concentrated from CCF by separating DNA, RNA, lipids, other cell and process derived components,

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Section: Resultsmentioning

confidence: 99%

Mass spectrometric evaluation of upstream and downstream process influences on host cell protein patterns in biopharmaceutical products

Falkenberg

Waldera-Lupa

Vanderlaan³

et al. 2019

Biotechnology Progress

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“…Full details regarding DSP microfabrication, operation, sample treatment efficiency, and comparison to other technologies for real‐time ESI‐MS have been described elsewhere. [ 19–21 ] As shown in Figure 2 , the DSP includes 1) a spatially resolved sampling interface for sterile, direct‐from‐culture media uptake, 2) an optimized “cross flow” sample treatment mass exchanger for inline sample preparation (see Supporting Information for details), and 3) an inline ESI emitter for online MS analysis. The DSP is a microfabricated mass exchanger with an integrated 360 µm OD, 50 µm ID PEEK (Upchurch Scientific) inlet for sample intake (probing tip), and a fused silica nano‐ESI emitter with a 360 µm OD and a 75 µm ID tapered to a 30 µm emitter (New Objective) outlet for inline ESI of the sample analytes for MS analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Simultaneously, chemicals which have been shown to enhance ESI‐MS performance (acetic acid or AA, and 3‐nitrobenzyl alcohol or m‐NBA) are injected into the sample channel to improve ionization and aid in MS detection of biomolecules. [ 19,20 ]…”

Section: Methodsmentioning

confidence: 99%

“…The mass ranges which were removed during winnowing are unlikely to have featured critical biomarkers because lower m/z ranges are not probed with DSP due to the removal of small molecules during sample treatment, while the high m/z range would contain contributions from high molecular weight media additives that are not part of cell secretome. [ 19,20 ]…”

Section: Methodsmentioning

confidence: 99%

“…[ 16–18 ] Recently, we demonstrated continuous ESI‐MS sensing with a ≈1 min response time for detecting the biomolecules serving as proxies to target CQA species. [ 19 ]…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Localized Sampling Enables Monitoring of Cell State via Inline Electrospray Ionization Mass Spectrometry

Chilmonczyk

Doron

Kottke

et al. 2020

Biotechnology Journal

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Nascent advanced therapies, including regenerative medicine and cell and gene therapies, rely on the production of cells in bioreactors that are highly heterogeneous in both space and time. Unfortunately, advanced therapies have failed to reach a wide patient population due to unreliable manufacturing processes that result in batch variability and cost prohibitive production. This can be attributed largely to a void in existing process analytical technologies (PATs) capable of characterizing the secreted critical quality attribute (CQA) biomolecules that correlate with the final product quality. The Dynamic Sampling Platform (DSP) is a PAT for cell bioreactor monitoring that can be coupled to a suite of sensor techniques to provide real‐time feedback on spatial and temporal CQA content in situ. In this study, DSP is coupled with electrospray ionization mass spectrometry and direct‐from‐culture sampling to obtain measures of CQA content in bulk media and the cell microenvironment throughout the entire cell culture process (≈3 weeks). Post hoc analysis of this real‐time data reveals that sampling from the microenvironment enables cell state monitoring (e.g., confluence, differentiation). These results demonstrate that an effective PAT should incorporate both spatial and temporal resolution to serve as an effective input for feedback control in biomanufacturing.

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Process analytical technologies in cell therapy manufacturing: State‐of‐the‐art and future directions

Wang

Bowles-Welch

Yeago

et al. 2021

J Adv Manuf & Process

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Cell therapies have the potential to effectively treat and even cure complex, currently untreatable diseases with unprecedented success. The cell therapy industry has been growing rapidly since the Food and Drug Administration approval of the first product in 2017. Despite tremendous promise, there are significant and unique challenges that must be overcome to make cell therapy manufacturing reproducible, scalable, high‐quality, and cost‐effective. Discovery and implementation of critical quality attributes (CQAs) and critical process parameters (CPPs) to the complex cell therapy manufacturing processes is one such grand challenge for the field. The role of process analytical technologies (PATs) in CQA/CPP discovery and eventual in‐process, or at‐process monitoring to maintain consistent process and product quality, is indispensable. Here we discuss the major challenges and the strategic framework for optimizing process development and related PATs for various cell therapies, with a focus on upstream processes. We introduce relevant approaches, such as quality‐by‐design (QbD), and the implementation of PATs to enable QbD in current biomanufacturing processes. We examine state‐of‐the‐art PAT implementation on standard physicochemical parameters in biopharmaceutical operations and consider potential cell therapy‐related parameters that may be instrumental in overcoming the challenges of the current cell therapy manufacturing landscape. Current innovations applied to the field, such as high‐throughput and high‐dimensional analyses, machine learning, and novel sensor technologies, are also discussed. We conclude that advances in PATs are necessary to identify CQAs and CPPs, overcome limitations in current operating processes, reduce overall product cost, and significantly accelerate the translation of laboratory discoveries into commercialized cell therapy products.

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Dynamic mass spectrometry probe for electrospray ionization mass spectrometry monitoring of bioreactors for therapeutic cell manufacturing

Cited by 11 publications

References 47 publications

Mass spectrometric evaluation of upstream and downstream process influences on host cell protein patterns in biopharmaceutical products

Mass spectrometric evaluation of upstream and downstream process influences on host cell protein patterns in biopharmaceutical products

Localized Sampling Enables Monitoring of Cell State via Inline Electrospray Ionization Mass Spectrometry

Process analytical technologies in cell therapy manufacturing: State‐of‐the‐art and future directions

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