ProSIAmpler Applications and Supporting Resources

Automated Real-Time Titer Determination

  • Real-time monitoring of monoclonal antibody (mAb) titer during bioprocessing is critical for process control and optimization. Traditional titer analysis requires manual sampling, extensive sample preparation, and offline HPLC analysis, leading to significant delays in data availability. The ProSIAmpler system addresses these challenges by providing a fully automated, online solution for real-time titer monitoring.

  • Full Automation: Eliminates manual sampling, purification and analysis steps

    Cost Savings:

    o   Eliminates need for liquid handlers ($50,000-150,000)

    o   Reduces consumables costs significantly:

    §  Traditional: $63,710/year for 5000 samples ($12.74/sample: $12.50 for ProA resin filter tip + $0.20 for regular tips + $0.042/sample for well plates)

    §  ProSIAmpler: $15,000/year for 5000 samples ($3/sample for ProA column)

    o   Minimizes labor costs -- no well plate handling (>90% reduction in hands-on time)

    Sustainability:

    o   Eliminates ~500 well plates and 4,000 pipette tips annually

    o   Reduces cold storage energy usage

    o   Minimizes biohazardous waste

    Fast Results: Sample-to-result in <30 minutes versus hours for manual methods

    • ProSIAmpler sampling module

    • 250uL Clarification Cartridge (250CC) with 0.65 μm filtration capability for automated sample clarification

    • Dual syringe pump liquid handling unit featuring:

      • Multi-position stream selector valve

      • FIAlab Protein A column

      • UV/Vis spectrophotometer (210-800 nm)

      • Integrated fluidics control system (SIAsoft -- now 21 CFR Part 11 Compliant)

  • Protein titer monitoring represents a critical process analytical parameter that directly impacts:

    • Process development optimization

    • Manufacturing efficiency

    • Product yield

    • Process economics

    • Batch release decisions

    The automated system provides real-time titer monitoring capabilities for:

    • Monoclonal antibodies

    • Bispecific antibodies

    • Fc fusion proteins

    • Other therapeutic proteins

    Real-time titer data enables:

    • Feed rate optimization

    • Harvest time determination

    • Process parameter adjustment

    • Product quality prediction

    • Manufacturing consistency

  • 1.     ProSIAmpler draws sample from bioreactor through integrated filtration

    2.     Sample passes through 250CC for cell removal and clarification:

    • Flow rate: 0.1-2 mL/min

    • Pressure: Up to 20 psi

    • No centrifugation required

    • Automated backflush capability prevents clogging

    3.     Clarified sample transfer to Protein A column

  • 1.     Column Preparation:

    • FIAlab ProA (0.125 mL) equilibration with 20 mM sodium phosphate pH 7.0

    • Flow rate: 1 mL/min

    • Pressure limit: 40 PSI

    2.     Sample Loading:

    • Automated volume adjustment based on UV feedback

    • Typical loading: 50-400 μg mAb

    • Linear response up to 200 μg demonstrated

    • Real-time concentration monitoring

    3.     Washing and Elution:

    • Column wash: 20 mM sodium phosphate pH 7.0

    • Elution: pH 2.8 phosphate buffer

    • Real-time UV monitoring at 280 nm

    • Automated fraction collection

    4.     Data Processing:

    • Automated peak integration

    • Concentration calculation using standard curve

    • Real-time result reporting

Automated Multi-Attribute Method

  • Multi-Attribute Method (MAM) enables comprehensive characterization of therapeutic proteins through peptide mapping and LC-MS analysis. The ProSIAmpler system automates the complex sample preparation workflow while maintaining data quality comparable to manual methods.

  • Traditional MAM Workflow (5000 samples/year):

    • Consumables: $63,710 ($12.74/sample for ProA tips, regular tips, and plates)

    • Labor: 2500 hours ($125,000)

    • Total: $188,710

    ProSIAmpler MAM Workflow:

    • Consumables: $15,000 ($3/sample for ProA column)

    • Labor: 250 hours ($12,500)

    • Total: $27,500

    Annual Savings: $161,210

    • ProSIAmpler sampling module with filtration

    • 250CC for automated clarification

    • Advanced liquid handling unit featuring:

      • Dual syringe pumps

      • Multi-position valves

      • Temperature-controlled reaction chambers

      • LC-MS interface

  • Multi-attribute monitoring provides comprehensive characterization of protein therapeutics by tracking:

    Critical Quality Attributes:

    • Primary sequence verification

    • Post-translational modifications

    • Sequence variants

    • Truncation variants

    • Structural modifications

    Post-Translational Modifications:

    • N-glycosylation patterns

    • O-glycosylation sites

    • Phosphorylation

    • Acetylation

    • Other chemical modifications

  • 1.     ProSIAmpler draws sample from bioreactor through integrated filtration

    2.     Sample passes through 250CC for cell removal and clarification:

    • Flow rate: 0.1-2 mL/min

    • Pressure: Up to 20 psi

    • No centrifugation required

    • Automated backflush capability prevents clogging

    3.     Clarified sample transfer to Protein A column

  • 1.     Column Preparation:

    • FIAlab ProA (0.125 mL) equilibration with 20 mM sodium phosphate pH 7.0

    • Flow rate: 1 mL/min

    • Pressure limit: 40 PSI

    2.     Sample Loading:

    • Automated volume adjustment based on UV feedback

    • Typical loading: 50-400 μg mAb

    • Linear response up to 200 μg demonstrated

    • Real-time concentration monitoring

    3.     Washing and Elution:

    • Column wash: 20 mM sodium phosphate pH 7.0

    • Elution: pH 2.8 phosphate buffer

    • Real-time UV monitoring at 280 nm

    • Automated fraction collection

    4.     Data Processing:

    • Automated peak integration

    • Concentration calculation using standard curve

    • Real-time result reporting

  • 1.     Denaturation/Reduction/Alkylation:

    • 6M guanidine HCl

    • DTT reduction at 37°C

    • Iodoacetamide alkylation

    • Dark reaction chamber

    2.     Buffer exchange:

    • Automated desalting

    • Salt removal verification

    3.     Enzymatic Digestion:

    • 37°C reaction chamber

  • For all applications, the ProSIAmpler system features:

    Sampling Module:

    • Programmable intervals: 0.1-1440 minutes

    • Sample volume: 100-2000 μL

    • Integrated 0.65 μm filtration

    • Automated cleaning cycles

    250CC Specifications:

    • Working volume: 250 μL

    • Flow rate: 0.1-2 mL/min

    • Maximum pressure: 20 psi

    • Material: modified Polyethersulfone (mPES), polysulfone and polyetheretherketon (PEEK)

    Liquid Handling:

    • Syringe resolution: 24,000 steps

    • Flow rate precision: ±1%

    • Volume accuracy: ±1%

    • Carryover: <0.1%

    Temperature Control:

    • Range: 4-95°C

    • Stability: ±0.1°C

    • Multiple independent zones

Automated Glycosylation Analysis

  • Glycosylation is one of the most critical quality attributes for biotherapeutic products, as it significantly impacts various factors such as protein folding and stability, pharmacokinetics and half-life, immunogenicity, biological activity, and product clearance rates. The ProSIAmpler platform addresses the need for efficient glycosylation monitoring by automating the process, seamlessly integrating sampling, deglycosylation, labeling, and analysis steps.

  • Traditional Workflow (5000 samples/year):

    • Consumables: $63,710 ($12.74/sample for ProA tips, regular tips, and plates)

    • Labor: 2000 hours ($100,000 at $50/hour)

    • Total: $163,710

    ProSIAmpler Workflow:

    • Consumables: $15,000 ($3/sample for ProA column)

    • Labor: 200 hours ($10,000)

    • Total: $25,000

    Annual Savings: $138,710

    • ProSIAmpler sampling module with integrated filtration

    • 250CC for automated clarification

    • Dual syringe pump liquid handling unit with:

      • Multi-position valves

      • Temperature-controlled reaction chambers

      • PGC enrichment column

      • HILIC-UPLC interface

  • This method provides automated, real-time monitoring of N-linked glycosylation patterns during bioprocess production. The system is compatible with various therapeutic proteins including:

    • Monoclonal antibodies

    • Fc fusion proteins

    • Other glycosylated biologics

  • 1.     ProSIAmpler draws sample from bioreactor through integrated filtration

    2.     Sample passes through 250CC for cell removal and clarification:

    • Flow rate: 0.1-2 mL/min

    • Pressure: Up to 20 psi

    • No centrifugation required

    • Automated backflush capability prevents clogging

    3.     Clarified sample transfer to Protein A column

  • 1.     Column Preparation:

    • FIAlab ProA (0.125 mL) equilibration with 20 mM sodium phosphate pH 7.0

    • Flow rate: 1 mL/min

    • Pressure limit: 40 PSI

    2.     Sample Loading:

    • Automated volume adjustment based on UV feedback

    • Typical loading: 50-400 μg mAb

    • Linear response up to 200 μg demonstrated

    • Real-time concentration monitoring

    3.     Washing and Elution:

    • Column wash: 20 mM sodium phosphate pH 7.0

    • Elution: pH 2.8 phosphate buffer

    • Real-time UV monitoring at 280 nm

    • Automated fraction collection

    4.     Data Processing:

    o   Automated peak integration

    o   Concentration calculation using standard curve

    o   Real-time result reporting

  • Standard Workflow

    1.     Protein denaturation:

    • 0.1% SDS/40 mM DTT in 50 mM Tris buffer

    • 95°C for 5 minutes

    • Volume: 100 µL

    • Final protein concentration: ~1 mg/mL

    2.     PNGase F digestion:

    • 0.5% NP-40 addition

    • Room temperature incubation

    • 5-60 minute protocols

    • 2 µL PNGase F (500,000 units/mL)

    • Enzyme:substrate ratio 1:20

    3.     2-AB labeling:

    • 48 mg/mL 2-AB in 85:15 DMSO:acetic acid

    • 1M 2-picoline borane (reducing agent)

    • 65°C for 2 hours

    • Equal volumes of sample and labeling solution

    • Final reaction volume: 100 µL

    4.     Sample cleanup:

    • 100 mg PGC column

    • Wash: 5% ACN + 0.1% TFA

    • Elution: 90% ACN + 0.1% TFA

    • 95% labeled glycan recovery

  • 1.     Combined denaturation and deglycosylation:

    • Instant denaturation buffer (proprietary Agilent formulation)

    • Rapid PNGase F enzyme

    • 50°C for 5 minutes

    • Single-step reaction

    2.     Instant labeling:

    • Instant PC fluorescent tag (proprietary Agilent formulation)

    • Room temperature

    • 5 minutes

    • No reduction step needed

    3.     Direct analysis:

    • No cleanup required

    • Immediate HILIC injection

    • Compatible with online analysis