Downstream Processing (DSP): Microscale Automation of Biopharmaceutical Processes Michel Eppink, Synthon BV, Nijmegen, The Netherlands
Overview Introduction Why automation? Automation in DSP processes Labscale Process Development Sophisticated Software Programs Process Analytical Technologies Conclusion
Introduction Automation of DSP processes is a slow process (conservative) Most of the work occurred in past years by own/common knowledge DSP processes consists of different unit operations and is not a continuous process The complexity of both the processes and the biomolecules faces a challenge for automation
Why Automation? Faster development of DSP processes Preventing human errors Structured approach Good electronic data collection
Automation in DSP Processes (protein purifications)
What are Proteins?
Proteins are complex molecules Interactions β-sheets protein α-helices
Proteins are complex molecules Primary structure (amino acids) Secondary structures (α-helices, β-sheets) Posttranslational modifications (e.g. glycans) Different interactions (hydrophobic, electrostatic, hydrogen bounding, van der Waals forces) keep the protein in a native energetically optimal structure Behavior in an ph, salt, additives environment determines the charged state and the formation of product related substances Purification strategies are a challenge, how do we use automation in the different processes?
Purification Development of proteins from small to large scale
Principle of a Purification Step Ligand: -Ionic -Hydrophobic -Affinity Column with Resin Chromatogram Sample mau BKA0023 IMAC003:10_U V1_280nm BKA0023 IMAC003:10_Logbook 4000 3000 2000 1000 Flow through Wash Elution Equilibration 0 sample injection Start washing Wash 2 Wash 3 Start elution strip 1M NaOH 300 400 500 600 ml Flow through Wash Elution
From small to large scale HTS 0.01 0.2 ml Scouting Studies 1-10 ml Research Run 200-300 ml Development Run 1-10 % (1-10 liter) Large Scale 100% (10-500 liter)
Small Large Experimental space
Labscale Process Development Robotic technologies Microfluidic technologies Sophisticated software programs Process analytical technologies
Robotic Technologies
Robotic Technologies Freedom EVO LiHa / RoMa Tecan Reader for result analysis Hotels/ carriers sample and buffer preparation Purification development occurs more and more with robotics Te-Chrom Te-Stack optional, for collection of fractions
Microtiterplate Studies 96-Position Filter Plates Batchwise chromatography (96/384 experiments /day) Titer (ELISA) HCP (ELISA) Protein UV Biacore assay MS (MALDI-TOF) SDS-PAGE Robotic Liquid Handling Screening of Resins Low/medium resolution No flow/bedheight properties map
Robotic Column Studies I Pre-packed with the resin of choice per each row Bed heights: 2.5, 5, 10, 30 mm; inner diameter: 5 mm Column volumes: 50, 100, 200, 600 µl
Robotic Column Studies II Sample loading Collection of fractions
Robotic Column Studies III Sample preparation/ clarification Preparation of Elution Buffers Sample Loading Elution Collect Fractions Collect Fractions Analysis Analysis (Tecan (UVReader) 280 )
Robotic Column Studies IV Chromatograms for the separation of two proteins (lysozyme, cytochrome c (each 1 mg/ml) on cation exchangers packed in 200µl Atoll columns for 8 RoboColumns using 0.1 M NaCl or 1 M NaCl respectively, processed on a common LC instrumentation or Robotics respectively xe+3 OD 280 mau nm [mau] 2 1 Shift due to lower dead volume 0 0 5 10 15 (min) Time [min] Chromatograms from a common LC instrumentation and Freedom EVO are identical Extremely high reproducibility from column to column Data kindly provided by TimSchroeder, Atoll, Germany
Microfluidic Technologies
Microfluidic Technologies Characterization of the biomolecules occurs gradually with microfluidic technologies in a high throughput fashion on: Content (concentration) Stability (melting temperature) Sugar content (heterogeneity) Aggregation (oligomers) Binding studies (Protein-Protein interactions)
Microfluidics Technologies Aggregation Content/Sugars Melting Temperature Binding studies
LabChip Analysis I => based on capillary electrophoresis Applications Purification development Determination of critical quality attributes Titer Purity Degree of glycosylation Fragmentation and Ab assembly Covalent aggregates
LabChip Analysis II <= The expected proteins are displayed as an electropherogram <= Sizing, concentration and purity results
Sophisticated Software Programs
Sophisticated Software Programs (Purification Process Development) Software program includes: Real-time flow scheme Trend curve data Display of all monitor values Method Logbook for full documentation Method start protocol Note books for pre/post-run and run notes Method handling with full flexibility Software used for both R&D and Production phases, no translations needed Complies with 21 CFR Part 11
Sophisticated Software Programs 2.0 1.7 Fractions 1 2 3 5 6 7 8 9 11 13 15 17 20 22 24 26 28 30 32 34 36 39 41 44 4648 50 53 56 58 100.0% Elution 250. 200. 1.5 Typical Chromatogram of a purification step 1.2 1.0 0.7 0.5 75. 50. 25. Regeneration 1 Reg 2 150. 100. 50. 0.2 Sample Load wash Reg 3 0.0 0. 0. - AU 00:00: 01:00: 02:00: Hr:Min: ms/c
Process Analytical Technologies
MAB-BUF J5 MAB-BUF J4 MAB-BUF J3 MAB-BUF J2 MAB-BUF J1 Cl. Harvest P-6 / V-103 Storage tank P-1 / C-101 CAPTURE S-107 S-126 Typical DSP process S-101 S-117 P-5 / C-103 AEX Capture and Virus Inactivation (BF building) ph adj. (J3) PW B1 1 M TRIS AEX-BUF ST AEX-BUF CIP AEX-BUF PS AEX-BUF M1 P-2 / V-101 S-118 Virus inactivation (LPH) S-125 S-106 S-124 S-102 P-15 / DE-102 0.22 µm Filtration S-121 P-13 / DE-101 S-119 0.45/0.22 µm Filtration Purification (HPLC) S-114 S-105 S-123 P-14 / V-102 Load conditioning S-120 LC-BUF M1 LC-pH adj. SuperPro Designer (Intelligen) S-104 S-122 P-3 / C-102 CEX CEX-BUF ST CEX-BUF CIP CEX-BUF PS CEX-BUF L2 CEX-BUF L1 S-103 S-108 S-116 S-128 VF-BUF O1 S-131 S-132 DF-BUF 01 S-112 S-134 S-135 Final Filling (Cleanroom) S-109 S-110 P-11 / DE-103 S-129 0.1 µm Filtration P-7 / DE-104 Virus Filtration S-111DF-FLUSH O1 P-8 / V-104 DF-BUF CIP S-130 Storage tank P-4 / DF-101 DF-BUF ST S-133 Ultrafiltration/Diafiltration S-127 S-115 S-113 DS-102 DS-101 P-12 / DE-105 S-136 0.22 µm Filtration P-9 / FL-101 P-10 Filling Truck (Discrete) DS-103
Integration of PAT Technologies Filtration Column steps Vessels Detection: 1)Absorbance 2)Conductivity 3)pH 4)Flow 5)Pressure 6)Temperature
Conclusions Automation has found gradually its way in DSP Microfluidics/Robotics are very useful and able to speed up the processes Software used will be implemented from start of development up to production PAT is still a minor part but will increase during coming years in DSP
Questions?