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Downstream Processing in Biopharmaceutical Manufacturing
Harvest and ClarificationTangential Flow Filtration (UF/DF)
Low Pressure Liquid Column Chromatography
Know the Characteristics of Your Protein Human Serum Albumin
Sequence of Amino AcidsMKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGEEHFKGLVLIA FSQYLQQCPFDEHVKLVNEL TEFAKTCVADESHAGCEKSL HTLFGDELCK VASLRETYGMADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEFKADEKKFWGKYLYEIARRHP YFYAPELLYYANKYNGVFQE CCQAEDKGACLLPKIETMRE KVLTSSARQR LRCASIQKFG ERALKAWSVA RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDRADLAKYICDN QDTISSKLKECCDKPLLEKS HCIAEVEKDAIPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKLKHLVDEPQNL IKQNCDQFEKLGEYGFQNAL IVRYTRKVPQVSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETYVPKAFDEKLF TFHADICTLPDTEKQIKKQT ALVELLKHKPKATEEQLKTV MENFVAFVDK CCAADDKEACFAVEGPKLVWSTQTALA
Tertiary Structure
Know the Characteristics of Your Protein
Human Serum Albumin:• MW (molecular weight = 69,000 Daltons (69 kD)• pI (isoelectric point) = 5.82• Hydropathicity (=hydrophobicity) = -.395
Typical Production Process Flow
(Feed 2)
(Feed 3)
(Feed 4)
Chrom 1Chrom 3
Cryo-preservation
Concentration / Diafiltration
Centrifuge
Viral Removal Filtration
(Feed1)Inoculum Expansion(Spinner Bottles)Ampule Thaw
Chrom 2
Media PrepMedia Prep
Working Cell Bank
Working Cell Bank
Sub- Culture
Sub- Culture
Inoculum
Sub- Culture
Sub- Culture Sub-
Culture
Sub- Culture Sub-
Culture
Sub- Culture Sub-
Culture
Sub- Culture
Large Scale Bioreactor
Wave Bag
Wave Bag
Seed Bioreactors
Fermentation
150L Bioreactor
750L Bioreactor
5,000L Bioreactor
26,000L Bioreactor
Depth Filtration
Depth Filtration
CollectionCollection
CentrifugeCentrifuge
Harvest/Recovery
HarvestCollection
Tank
1,500L
HarvestCollection
Tank
1,500L
FilterChromatography
Skid
Anion Exchange Chromatography (QXL)
ColumnEluateHold Tank
8,000L
EluateHold Tank
8,000L
EluateHold Tank
6,000L
EluateHold Tank
6,000L
FilterChromatography
Skid
Protein A Chromatography
Column
Chromatography Skid
Column
EluateHold Tank
20,000L
EluateHold Tank
20,000L
Hydrophobic Interaction Chromatography (HIC)
EluateHold Tank
20,000L
EluateHold Tank
20,000L
Viral Inactivation
EluateHold Tank
5,000L
EluateHold Tank
5,000L
FilterChromatography
Skid
Anion Exchange Chromatography
(QFF - Fast Flow)
Column
Post-viralHold
Vessel3,000L
Post-viralHold
Vessel3,000L
Viral Filtering Ultra FiltrationDiafiltration
Bulk Fill
Purification
24 days 31 days
8 days
1 dayLSCC Mfg Process Overview
Clarification or Removal of Cells and Cell Debris
Using Centrifugation(Using Depth Filtration)
Continuous CentrifugationMedia and Cells In & Clarified Media Out
Separation of particles from liquid by applying a pressure to the solution to force the solution through a filter. Filters are materials with pores.
Particles larger than the pore size of thefilter are retained by the filter.
Particles smaller than the pore size of the filter pass through the filter along with the liquid.
Filtration
Uses crossflow to reduce build up of retained components on the membrane surface
Allows filtration of high fouling streams and high resolution
Tangential Flow Filtration
Tangential Flow Filtration – TFFSeparation of Protein of Interest
Using TFF with the right cut off filters, the protein of interest can be separated from other proteins and molecules in the clarified medium.
HSA has a molecular weight of 69KD. To make sure that the protein of interest is retained, a 10KD cut-off filter is used.
After we concentrate or ultrafilter our protein, we can diafilter, adding the phosphate buffer at pH 7.1 that we will use to equilibrate our affinity column to prepare for affinity chromatography of HSA.
Overview of TFF SOP• Prepare buffer:
Sodium phosphate buffer pH 7.1• Set up the apparatus-CAUTION Stored in NaOH• Flush with water-CAUTION Stored in NaOH
Adjust flow rate to 30-50ml/minFlush retentate lineFlush permeate line
• Precondition with buffer (just the permeate line)
• Perform TFF• Prepare cleaning solution (NaOH)• Flush with water• Flush with NaOH to clean and store
Downstream Processing Equipment
Lab-Scale TFF System Large-Scale TFF System
Lab-Scale TFF Filter = Pall’s Pellicon
How TFF Concentrates and Diafiltersthe Protein of Interest
Low Pressure Production Chromatography
The System: Components and ProcessesThe Media: Affinity, Ion Exchange,
Hydrophobic Interaction Chromatography and Gel Filtration
LP LC Components
• Mixer for Buffers, Filtrate with Protein of Interest, Cleaning Solutions
• Peristaltic Pump• Injector to Inject Small Sample (in our case for
HETP Analysis)• Chromatography Column and Media (Beads)• Conductivity Meter• UV Detector
Peristaltic Pump
• Creates a gentle squeezing action to move fluid through flexible tubing.
UV Detector
Detects proteins coming out of the column by measuring absorbance at 280nm
Conductivity Meter
• Measures the amount of salt in the buffers – high salt or low salt are often used to elute the protein of interest from the chromatography beads.
• Also measures the bolus of salt that may be used to determine the efficiency of column packing (HETP)
Liquid Column Chromatography ProcessPurge Air from System with Equilibration BufferPack Column with Beads (e.g. ion exchange,
HIC, affinity or gel filtration beads)Equilibrate Column with Equilibration BufferLoad Column with Filtrate containing Protein of
Interest in Equilibration Buffer Wash Column with Equilibration BufferElute Protein of Interest with Elution Buffer of
High or Low Salt or pHRegenerate Column or Clean and Store
Downstream Processing EquipmentLab Scale Chromatography System
Large Scale Chromatography System
Overview of LP LC Chromatography• The molecules of interest, in our case proteins, are adsorbed or
stuck to beads packed in the column. We are interested in the equilibrium between protein free in solution and protein bound to the column. The higher the affinity of a protein for the bead the more protein will be bound to the column at any given time. Proteins with a high affinity travel slowly through the column because they are stuck a significant portion of the time. Molecules with a lower affinity will not stick as often and will elute more quickly. We can change the relative affinity of the protein for the column (retention time) and mobile phase by changing the mobile phase (the buffer). Hence the difference between loading buffers and elution buffers. This is how proteins are separated.
• The most common type of adsorption chromatography is ion exchange chromatography. The others used in commercial biopharmaceutical production are affinity, hydrophobic interaction and gel filtration.
Column Chromatography Separates molecules by their chemical and
physical differences Most common types:
Size exclusion (Gel filtration): separates by molecular weight
Ion exchange: separates by charge Affinity chromatography: specific binding Hydrophobic Interaction: separates by
hydrophobic/hydrophilic characteristics
Gel Filtration Chromatography
Ion Exchange Chromatography Ion Exchange Chromatography relies on charge-charge interactions between the protein of interest and charges on a resin (bead). Ion exchange chromatography can be subdivided into cation exchange chromatography, in which a positively charged protein of interest binds to a negatively charged resin; and anion exchange chromatography, in which a negatively charged protein of interest binds to a positively charged resin.One can manipulate the charges on the protein by knowing the pI of the protein and using buffers of different pHs to alter the charge on the protein. Once the protein of interest is bound, the column is washed with equilibration buffer to remove unattached entities. Then the bound protein of interest is eluted off using an elution buffer of increasing ionic strength or of a different pH. Either weakens the attachment of the protein of interest to the bead and the protein of interest is bumped off and eluted from the resin.Ion exchange resins are the cheapest of the chromatography media available and are therefore almost always used as a step in biopharmaceutical protein production purification.
Isoelectric Focusing or IEFOnce you know the pI of your
protein (or the pH at which your protein is neutral), you can place it in a buffer at a lower or higher pH to alter its charge. If the pH of the buffer is less than the pI, the protein of interest will become positively charged. If the pH of the buffer is greater than the pI, the protein of interest will become negatively charged.
pH < pI < pH + 0 -
Affinity ChromatographyAffinity chromatography separates the protein of interest on the basis of a reversible interaction between it and its antibody coupled to a chromatography bead (here labeled antigen) .With high selectivity, high resolution, and high capacity for the protein of interest, purification levels in the order of several thousand-fold are achievable.The protein of interest is collected in a purified, concentrated form. Biological interactions between the antigen and the protein of interest can result from electrostatic interactions, van der Waals' forces and/or hydrogen bonding. To elute the protein of interest from the affinity beads, the interaction can be reversed by changing the pH or ionic strength.The concentrating effect enables large volumes to be processed. The protein of interest can be purified from high levels of contaminating substances.Making antibodies to the protein of interest is expensive, so affinity chromatography is the least economical choice for production chromatography.
Time (min)
Abs 280nm
Affinity Chromatography
Hydrophobic Interaction Chromatography (HIC)
HIC is finding dramatically increased use in production chromatography. Antibodies are quite hydrophobic and therapeutic antibodies are the most important proteins in the biopharmaceutical pipeline. Since the molecular mechanism of HIC relies on unique structural features, it serves as a non-redundant option to ion exchange, affinity, and gel filtration chromatography. It is very generic, yet capable of powerful resolution. Usually HIC media have high capacity and are economical and stable. Adsorption takes place in high salt and elution in low salt concentrations.
Component Culture Harvest Level Final Product Level Conventional Method
Therapeutic Antibody 0.1-1.5 g/l 1-10 g/l UF/Cromatography
Isoforms Various Monomer Chromatography
Serum and host proteins 0.1-3.0 g/l < 0.1-10 mg/l Chromatography
Cell debris and colloids 106/ml None MF
Bacterial pathogens Various <10-6/dose MF
Virus pathogens Various <10-6/dose (12 LRV) virus filtration
DNA 1 mg/l 10 ng/dose Chromatography
Endotoxins Various <0.25 EU/ml Chromatography
Lipids, surfactants 0-1 g/l <0.1-10 mg/l Chromatography
Buffer Growth media Stability media UF
Extractables/leachables Various <0.1-10 mg/l UF/ Chromatography
Purification reagents Various <0.1-10mg/l UF
Common Process Compounds and Methods of Purification or Removal
Actual BioLogic System
• Complex System• Not easy to ‘see’
interaction of components• Students use virtual
system to prepare to use actual system
• Use virtual system for BIOMANonline
• System same as industrial chromatography skid
Conductivity Meter
UV Detector
Mixer
PeristalticPump
Column
Injector Valve
Buffer Select
The Virtual Chromatography Laboratory
http://ATeLearning.com/BioChrom/