Advanced analytical technologies for rapid screening of process and product quality attributes supporting upstream bioprocessing

Significance of the topic

Rapid, robust analytics are critical in upstream bioprocess development to accelerate the design-make-test cycle and enable data-driven process optimization. High-throughput, automatable assays for titer, aggregation, intact and reduced protein forms, released glycans, and media/metabolite profiling allow process engineers to receive timely feedback from parallel bioreactor experiments (e.g., Ambr250). Integrating automated sample preparation, short LC/LC–MS methods, and streamlined data visualization improves experiment throughput, reduces sample consumption, and shortens decision timelines for cell line and process development.

Objectives and study overview

The work presents a suite of rapid screening methods and automated workflows implemented at Waters Immerse Delaware to support upstream bioprocessing. Objectives were to: (1) develop short-run LC and LC–MS assays for key product quality attributes (PQAs) and media/metabolites, (2) implement automated sample preparation using Andrew+ pipetting robots and ProA affinity capture, (3) apply methods to Ambr250 DOE bioreactor experiments, and (4) enable efficient data handling and visualization via a JMP add-in.

Used instrumentation

Key instrumentation and automation described and used:

• Ambr® 250 multi-parallel bioreactor system (Sartorius) for upstream DOE experiments.
• Andrew+ Pipetting Robots for automated sample handling, ProA capture, and released glycan preparation.
• ACQUITY UPLC (Premier) systems and BioAccord high-resolution LC–MS platform (Waters) for chromatographic and MS analyses.
• Columns: ACQUITY Premier HSS T3 (RP, 2.1 x 100 mm, 1.8 μm) for media/metabolites; ProA affinity column (2.1 x 20 mm) for titer; ACQUITY Premier SEC 250 Å (4.6 x 100 mm and 4.6 x 300 mm) for aggregation screening and characterization; ACQUITY Premier Protein BEH C4 300 Å (2.1 x 50 mm) for intact and reduced protein analyses; ACQUITY Premier Glycan BEH Amide 130 Å (2.1 x 150 mm) for released glycan profiling.
• Supporting bioanalyzers for cell counting and additional in-process measurements.

Methodology

Automated sample preparation and small-volume workflows

Samples consisted of clarified spent media (raw) and ProA affinity-purified samples. Andrew+ robots handled ProA capture, denaturation/reduction for LC analysis, and released glycan kit workflows; total per-sample consumption examples included ~150 µL of spent media and ~70 µL of ProA-purified material across assays, demonstrating low-volume compatibility for high-throughput screens.

Media and metabolite profiling (LC–MS)

Reversed-phase LC–HRMS on ACQUITY Premier HSS T3 with a 9 min runtime (positive and negative polarity runs, 2 µL injection). Method uses a compound library (220+ analytes) and waters_connect guided workflow. Mobile phases: A = 0.1% formic acid (aqueous), B = 90% ACN/10% IPA/0.1% FA. Typical MS settings: desolvation ~550 °C, capillary voltage ~1 kV (pos), cone 20 V (pos). The assay yields total ion chromatograms (TIC), extracted ion chromatograms (XICs) for metabolites (e.g., glutamate), and temporal trends across bioreactor runs.

Titer by ProA affinity LC–UV

ProA affinity column (2.1 x 20 mm) on ACQUITY Premier LC–UV, 3 min runtime, injection 1–2 µL, column at 25 °C. Mobile phase A = 1× DPBS, B = 0.1% formic acid; detection at 280 nm. Protocol includes gradient cycling post-elution to minimize carryover. Assay quantifies mAb titer directly from clarified spent media after minimal preparation.

Aggregation by SEC (screening and characterization)

Two SEC modalities: a rapid screening method using a 4.6 x 100 mm Premier SEC (1.7 µm) with isocratic 1× DPBS at 0.5 mL/min, 4 min runtime (no dilution for clarified media or ProA purified samples); and a characterization method using 4.6 x 300 mm column, 0.3 mL/min, 15 min runtime on ProA-purified samples. Detection at 280 nm to quantify monomer versus aggregates.

Intact and reduced protein LC–MS analyses

Intact protein: ACQUITY Premier Protein BEH C4 (2.1 x 50 mm), BioAccord LC–MS, 5 min run, samples ProA purified and diluted 1:10 in formic acid, high column temp (80 °C), MS settings optimized for intact mass detection (desolvation 550 °C, capillary 1.5 kV, cone 70 V). Deconvolution of observed spectra yields intact mass distributions and major proteoforms.

Reduced protein: denaturation with guanidine and DTT reduction to generate heavy chain (HC) and light chain (LC) fragments, analyzed on same BEH C4 column with 5 min runtime (column temp 60 °C), MS parameters adjusted for subunit mass detection. This enables rapid monitoring of HC/LC mass variants and deglycosylation or clipping events.

Released glycan analysis

Released glycans prepared with Waters glycan kits on Andrew+ robots, analyzed on ACQUITY Premier Glycan BEH Amide (2.1 x 150 mm) using HILIC separation, 10 min runtime. Mobile phases: ammonium formate glycan buffer (A) and ACN (B). MS settings tuned for glycan detection; results include TIC and identification of major glycan species.

Main results and discussion

Method performance and throughput

The portfolio of assays delivers rapid, low-volume analytics compatible with parallel bioreactor workflows. Typical runtimes are short (3–15 min depending on assay), enabling frequent sampling and fast turnaround. Sharing common mobile phase chemistries across several assays (e.g., water/0.1% FA and 90% ACN/10% IPA/0.1% FA) simplifies buffer preparation and instrument setup. ProA capture enables direct transfer of product-focused assays (intact, reduced, glycan, SEC characterization) from spent media with minimal matrix effects.

Representative outputs and utility

Examples shown include TICs and XICs for metabolites with trending across 14-day Ambr runs, LC–UV ProA chromatograms for titer trends, SEC profiles for aggregate screening and characterization, deconvoluted intact and reduced mass spectra showing proteoform distributions, and glycan profiles indicating major glycoforms. The JMP add-in enables organized import and visualization (overlaid trends across bioreactors, pathway-focused grouping), facilitating rapid interpretation of multi-assay datasets.

Benefits and practical applications

• High-throughput screening: Short run times and low sample volumes permit dense temporal monitoring across many bioreactors during DOE studies.
• Automation: Andrew+ integration reduces manual prep, improves reproducibility, and supports standardized ProA capture and glycan workflows.
• Flexibility: Combination of screening and characterization modes (e.g., SEC short vs. long columns) balances speed and resolution depending on decision stage.
• Data integration: JMP add-in simplifies data aggregation and visualization critical for process optimization decisions.

Future trends and potential applications

• Tighter integration of automation, LIMS, and real-time analytics could enable closed-loop design-of-experiment adjustments and richer process understanding.
• Expanded libraries for media/metabolite LC–MS and improved semi-quantitative workflows will support metabolic phenotyping of cell culture conditions at scale.
• Miniaturized or multiplexed LC–MS platforms and faster columns may further reduce cycle times and increase bioreactor sampling frequency.
• Application to newer modalities (bispecifics, ADCs) will require adaptation of capture chemistries and MS methods but can leverage the same automated preparative workflows.

Conclusion

The presented analytical toolkit demonstrates that automated sample preparation combined with rapid LC and LC–MS assays provides timely, actionable data for upstream bioprocessing. Implemented on an Ambr250 DOE platform, these methods enable frequent monitoring of titer, aggregation, intact/reduced proteoforms, released glycans, and media metabolites with low sample consumption. Standardized mobile phases and automation simplify lab workflows and accelerate decision-making in cell line and process development.

References

• Poster: Advanced analytical technologies for rapid screening of process and product quality attributes supporting upstream bioprocessing; Waters Immerse Delaware. Authors: Yun Alelyunas, Richard Jeske, Julie Wushensky, Chandler Przybylski, Jurgen Sanes. Document ref: 720009578EN. NIIMBL-Confidential.