Downstream Process Validation

• Downstream processing is the separation of product from other culture medium components (purification)
• Downstream processing is sometimes very time-consuming and costly
• Purification is necessary because other medium components may interfere with protein function or cause adverse reaction if protein is introduced into a human
• Protein Purification


  • High-resolution chromatographic purification is usually undertaken
  • A combination of 2-4 different chromatographic techniques is used in a typical downstream processing procedure
  • Affinity chromatography is used wherever possible due to its high biospecificity which results in a very high degree of purification
  • High resolution chromatography normally yields a protein that is 98-99% pure
• Downstream Processing


  • Normally carried out under clean room conditions
  • The final steps (e.g. sterile filtration and aseptic filling into final product containers) are carried out under Grade A laminar flow conditions
• Considerations when selecting a purification strategy


  • Keep the purification simple, so it can be easily reproduced – the less steps the better
  • Keep it cheap
  • Adopt a step approach and optimise each step along the way
  • Speed is important so avoid slow techniques and delays
  • Use reliable and proven techniques and equipment
• Objectives when selecting a purification strategy

  • High yield
  • High purity
  • High product stability
  • Scale of operation
  • Reproducibility
  • Economical use of reagents/equipment
  • Convenience
• Clarification


  • Stands in the critical junction of Fermentation and further Downstream processing
  • One of the most critical steps in biopharmaceutical production
  • Primary objective is separation of very low concentration of product from high percentage of contaminants
  • Directly affects product yield, consistency and reproducibility
• Common technologies of post fermentation clarification

  • Centrifugation
  • Tangential flow filtration
  • Depth filtration
  • Surface filtration
• Initial Product Recovery


  • Select extraction procedure according to source and location of protein
  • Use gentle procedures to minimise acidification and release of proteolytic enzymes
  • Work quickly at sub-ambient temperatures
  • Use buffer to maintain pH, ionic strength
• Harvesting of Cells


  • Harvesting steps should be performed in equipment and areas designed to minimize the risk of contamination
  • Harvest and purification procedures that remove or inactivate the producing organism, cellular debris and media components (while minimizing degradation, contamination, and loss of quality) should be adequate to ensure that the intermediate or product is recovered with consistent quality
• Intracellular Protein Recovery


  • If the target protein is intracellular, then it needs to be released from the cell
  • An extraction medium must be selected in which the target protein is stable
  • There are many methods available for cell disruption, the choice will be dependent on the nature of the cellular material and should always be as gentle as possible
• Disruption Methods


  • Freeze-thawing
  • Bead milling
  • Homogenisation
  • Blending/Grinding
  • Ultrasonication
  • Cell lysis with proteolytic enzymes
• Direct Flow Filtration (DFF)


  Also known as "dead-end" or 'normal flow' filtration, applies the feed stream perpendicular to the membrane face and attempts to pass 100% of the fluid through the membrane
• Tangential Flow Filtration (TFF)


  Also known as crossflow filtration, where the feed stream passes parallel to the membrane face as one portion passes through the membrane (permeate) while the remainder (retentate) is recirculated back to the feed reservoir
• Depth Filtration


  • A type of normal flow filtration
  • Porous medium capable of retaining particles, colloids throughout its width rather than just on the surface
  • The major advantages are: single use (reduction of process validation requirements), effective removal of contaminants, cost effectiveness (no CIP and SIP), scalable
• Depth Filtration Mechanisms


  • Mechanical sieving
  • Adsorption
• Tangential Flow Filtration (TFF) Theory


  • The flow of sample solution across the membrane surface sweeps away aggregating molecules that form a membrane-clogging gel (gel polarization), allowing molecules smaller than the membrane pores to move toward and through the membrane
  • TFF can be faster and more efficient than DFF for size separation
• TFF Validation


  • Verification that the molecular weight cut-off for ultrafiltration membranes is appropriate for the intended use
  • Verification that the pore rating for microfiltration membranes is appropriate for the intended use
  • Verification that the membrane used meets the specifications for the process step
  • Verification of critical process parameters such as filtrate and retentate pressures, fluxes, and temperatures
• TFF Validation for Protein Concentration or Buffer Exchange

  • The primary process parameters to measure include the operating ranges of pressure, flow, and temperature
  • For diafiltration, the removal of low molecular weight contaminants should also be monitored
  • The operating ranges under which the product does not undergo adverse changes, such as aggregation, denaturation, or loss of activity, should be defined
  • It may also be useful to investigate the effect of changes in flux on product recovery and activity
• TFF Validation for Cell Debris Removal / Clarification

  • If the TFF operation is only a clarification step, process qualification should address issues of permeate clarity, product quality, and yield
  • If the TFF process is actually a purification step, validation must also establish that the degree of purification is acceptable and consistent
  • The process should be tested at the extremes of the manufacturing operating ranges for tangential flow rates, pressures, and flux to verify that fouling is not occurring and does not adversely affect product quality
  • For the removal of specific contaminants, clearance studies should be conducted
• Validation of Filters


  • Filter compatibility is tested with process conditions to avoid nonspecific binding of product to the filter or addition of extractables to the process stream
  • Extractables & leachables are defined and limits established based on the final product safety studies
  • Special considerations apply for sterilising filters and those that are designed for virus removal
• Protein Purification Methods


  • Precipitation (solubility)
  • Size exclusion chromatography (mass/shape)
  • Ion-exchange / chromatofocusing (charge)
  • Hydrophobic interaction chromatography (hydrophobicity)
  • Affinity chromatography (specific binding)
  • Metal chelate chromatography (metal binding)
• Process Validation for Downstream Processing

  • Typically involves several chromatography and tangential flow filtration (TFF) steps
  • Chromatography steps provide product purification while the TFF steps are used for removing or concentrating macromolecules
  • Several chromatography steps are required to achieve the level of purity required for a protein to be used as a therapeutic agent
• Downstream Process Validation


  • Involves performance qualifications (PQ) that require rigorous testing to demonstrate the effectiveness and repeatability of the process
  • The goal of PQ is to establish confidence in the performance of purification unit operations under normal as well as 'worst-case' conditions at the extreme of normal operating conditions
• Downstream Processing Validation Stages

  • Process chemicals and raw materials
  • Column packing materials
  • Membranes for tangential flow filtration
  • Chromatography column packing
  • Clearance studies – viral, immunogenic, pyrogenic contaminants
  • Tangential flow filtration
• Validation of Column Chromatography

  • Isolation and purification of a protein is based upon its chemical and physical properties size, shape, charge, solubility
  • Proteins are fractionated by column chromatography as different proteins are retarded to different extents by their interaction with the matrix
• Common Chromatography Columns


  • Protein A
  • Ion exchange (AEX, CEX)
  • Hydrophobic interaction (HIC)
  • Affinity (AC)
  • Size exclusion/Gel filtration (SE/GF)
• Impurity clearance is evaluated through laboratory-scale studies and testing of in-process pools from conformance lots
• Removal of impurities that have the potential to adversely affect product safety or intended biological activity is validated
• More common columns in column chromatography

  • Protein A
  • IEX (Ion exchange chromatography)
  • HIC (Hydrophobic interaction column)
  • AC (Affinity chromatography)
  • SE/GF (Size exclusion/Gel filtration chromatography)
• Protein A


  Use of immobilized Protein A which binds Mab from the harvested cell culture fluid
• IEX (Ion exchange chromatography)


  Good for capture, intermediate, and polish. These can be either AEX (anion exchange) or CEX (cation exchange) chromatography
• HIC (Hydrophobic interaction column)


  Good for intermediate purification
• AC (Affinity chromatography)


  Good for capture and intermediate purification
• SE/GF (Size exclusion/Gel filtration chromatography)


  Good polishing step
• Hypothetical Operational Parameter Summary for Downstream Purification:

  Column load, flow rate, buffer composition, load/wash pH, load/wash conductivity, elution pH, elution conductivity, yield, purity
• Impurity clearance


  Evaluated through laboratory-scale studies and testing of in-process pools from conformance lots
• Impurity clearance


  Removal of impurities that have the potential to adversely affect product safety or intended biological activity to sufficiently low levels should be consistently demonstrated
• Initial scientific assessment


  Conducted to identify and select those impurities requiring study