Presentation on theme: "Downstream Processing in Biopharmaceutical Manufacturing"— Presentation transcript:
1 Downstream Processing in Biopharmaceutical Manufacturing Harvest and ClarificationTangential Flow Filtration (UF/DF)Low Pressure Liquid Column Chromatography
2 Know the Characteristics of Your Protein Human Serum Albumin Sequence of Amino AcidsTertiary StructureMKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPFDEHVKLVNEL TEFAKTCVAD ESHAGCEKSL HTLFGDELCK VASLRETYGMADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEFKADEKKFWGK YLYEIARRHP YFYAPELLYYANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLTSSARQR LRCASIQKFG ERALKAWSVA RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKECCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEKLGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLPDTEKQIKKQT ALVELLKHKP KATEEQLKTV MENFVAFVDK CCAADDKEACFAVEGPKLV WSTQTALA
3 Know the Characteristics of Your Protein Human Serum Albumin:MW (molecular weight = 69,000 Daltons (69 kD)pI (isoelectric point) = 5.82Hydropathicity (=hydrophobicity) =
4 Viral Removal Filtration Typical Production Process Flow(Feed 4)(Feed 2)Centrifuge(Feed1)Ampule ThawInoculum Expansion(Spinner Bottles)(Feed 3)Cryo-preservationConcentration /DiafiltrationChrom 2Chrom 1Chrom 3Viral Removal Filtration
5 LSCC Mfg Process Overview Large Scale BioreactorWaveBagSeed BioreactorsFermentation150L Bioreactor750L Bioreactor5,000L Bioreactor26,000L Bioreactor1 dayMedia PrepWorking CellBankSub-CultureInoculumDepthFiltrationCollectionCentrifugeHarvest/Recovery24 days31 daysHarvestCollectionTank1,500LFilterChromatography SkidAnion Exchange Chromatography (QXL)ColumnEluateHold8,000L6,000LProtein A Chromatography20,000LHydrophobic Interaction Chromatography (HIC)Viral Inactivation5,000LAnion Exchange Chromatography (QFF - Fast Flow)Post-viralVessel3,000LViral FilteringUltra FiltrationDiafiltrationBulk FillPurification8 days
6 Clarification or Removal of Cells and Cell Debris Using Centrifugation(Using Depth Filtration)
7 Continuous Centrifugation Media and Cells In & Clarified Media Out Continuous multichamber disc-stack centrifuge. The bowl contains anumber of parallel discs providing a large clarifying surface with a small sedimentation distance. The sludge (cells) is removed through
8 Filtration 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.
9 Tangential Flow Filtration Uses crossflow to reduce build up of retained components on the membrane surfaceAllows filtration of high fouling streams and high resolution
10 Tangential Flow Filtration – TFF Separation 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.
11 Overview of TFF SOP Prepare buffer: Sodium phosphate buffer pH 7.1 Set up the apparatus-CAUTION Stored in NaOHFlush with water-CAUTION Stored in NaOHAdjust flow rate to 30-50ml/minFlush retentate lineFlush permeate linePrecondition with buffer (just the permeate line)Perform TFFPrepare cleaning solution (NaOH)Flush with waterFlush with NaOH to clean and store
12 Downstream Processing Equipment Lab-Scale TFF SystemLarge-Scale TFF System
14 How TFF Concentrates and Diafilters the Protein of Interest
15 Low Pressure Production Chromatography The System: Components and ProcessesThe Media: Affinity, Ion Exchange, Hydrophobic Interaction Chromatography and Gel Filtration
16 LP LC ComponentsMixer for Buffers, Filtrate with Protein of Interest, Cleaning SolutionsPeristaltic PumpInjector to Inject Small Sample (in our case for HETP Analysis)Chromatography Column and Media (Beads)Conductivity MeterUV Detector
17 Peristaltic PumpCreates a gentle squeezing action to move fluid through flexible tubing.Creates a gentle squeezing action to move fluid through flexible tubing.In this example three rollers on rotating arms pinch the tube against an arc and move the fluid along. There are usually three or four sets of rollers
18 UV DetectorDetects proteins coming out of the column by measuring absorbance at 280nm
19 Conductivity MeterMeasures 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)
20 Liquid Column Chromatography Process Purge 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 BufferWash Column with Equilibration BufferElute Protein of Interest with Elution Buffer of High or Low Salt or pHRegenerate Column or Clean and Store
21 Downstream Processing Equipment Lab ScaleChromatography SystemLarge ScaleChromatography System
22 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.
23 Column Chromatography Separates molecules by their chemical and physical differencesMost common types:Size exclusion (Gel filtration): separates by molecular weightIon exchange: separates by chargeAffinity chromatography: specific bindingHydrophobic Interaction: separates by hydrophobic/hydrophilic characteristics23
25 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.
26 Isoelectric Focusing or IEF Once 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
27 Affinity Chromatography Affinity 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.
29 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.
30 Common Process Compounds and Methods of Purification or Removal ComponentCulture Harvest LevelFinal Product LevelConventional MethodTherapeutic Antibodyg/l1-10 g/lUF/CromatographyIsoformsVariousMonomerChromatographySerum and host proteinsg/l< mg/lCell debris and colloids106/mlNoneMFBacterial pathogens<10-6/doseVirus pathogens<10-6/dose (12 LRV)virus filtrationDNA1 mg/l10 ng/doseEndotoxins<0.25 EU/mlLipids, surfactants0-1 g/l< mg/lBufferGrowth mediaStability mediaUFExtractables/leachablesUF/ ChromatographyPurification reagents<0.1-10mg/l
31 Actual BioLogic System Complex SystemNot easy to ‘see’ interaction of componentsStudents use virtual system to prepare to use actual systemUse virtual system for BIOMANonlineSystem same as industrial chromatography skid