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Downstream Processing in Biopharmaceutical Manufacturing

Downstream Processing in Biopharmaceutical Manufacturing. Harvest and Clarification Tangential Flow Filtration (UF/DF) Low Pressure Liquid Column Chromatography. Know the Characteristics of Your Protein Human Serum Albumin. Sequence of Amino Acids. Tertiary Structure.

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Downstream Processing in Biopharmaceutical Manufacturing

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  1. Downstream Processing in Biopharmaceutical Manufacturing Harvest and Clarification Tangential Flow Filtration (UF/DF) Low Pressure Liquid Column Chromatography

  2. Know the Characteristics of Your Protein Human Serum Albumin Sequence of Amino Acids Tertiary Structure MKWVTFISLL 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) = 5.82 • Hydropathicity (=hydrophobicity) = -.395

  4. Concentration / Diafiltration (Feed 2) (Feed1) Centrifuge Chrom 1 Chrom 2 Cryo-preservation (Feed 4) Typical Production Process Flow Inoculum Expansion (Spinner Bottles) Ampule Thaw (Feed 3) Chrom 3 Viral Removal Filtration

  5. LSCC Mfg Process Overview Large Scale Bioreactor Media Prep Seed Bioreactors 26,000L Bioreactor Centrifuge Working Cell Bank 5,000L Bioreactor 750L Bioreactor 150L Bioreactor Depth Filtration Wave Bag Sub- Culture Sub- Culture Sub- Culture Sub- Culture Sub- Culture Collection Inoculum Fermentation Harvest/Recovery Viral Inactivation Eluate Hold Tank 8,000L Filter Column Harvest Collection Tank 1,500L Chromatography Skid Purification Eluate Hold Tank 20,000L Eluate Hold Tank 20,000L Anion Exchange Chromatography (QXL) Filter Column Eluate Hold Tank 6,000L Filter Column Chromatography Skid Column Chromatography Skid Eluate Hold Tank 5,000L Post-viral Hold Vessel 3,000L Chromatography Skid Protein A Chromatography Viral Filtering Anion Exchange Chromatography (QFF - Fast Flow) Ultra Filtration Diafiltration Bulk Fill Hydrophobic Interaction Chromatography (HIC) 1 day 24 days 31 days 8 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

  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 the filter 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 surface Allows filtration of high fouling streams and high resolution

  10. 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.

  11. 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/min Flush retentate line Flush 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

  12. Downstream Processing Equipment Lab-Scale TFF System Large-Scale TFF System

  13. Lab-Scale TFF Filter = Pall’s Pellicon

  14. How TFF Concentrates and Diafiltersthe Protein of Interest

  15. Low Pressure Production Chromatography The System: Components and Processes The Media: Affinity, Ion Exchange, Hydrophobic Interaction Chromatography and Gel Filtration

  16. 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

  17. Peristaltic Pump • Creates a gentle squeezing action to move fluid through flexible tubing.

  18. UV Detector Detects proteins coming out of the column by measuring absorbance at 280nm

  19. 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)

  20. Liquid Column Chromatography Process • Purge Air from System with Equilibration Buffer • Pack Column with Beads (e.g. ion exchange, HIC, affinity or gel filtration beads) • Equilibrate Column with Equilibration Buffer • Load Column with Filtrate containing Protein of Interest in Equilibration Buffer • Wash Column with Equilibration Buffer • Elute Protein of Interest with Elution Buffer of High or Low Salt or pH • Regenerate Column or Clean and Store

  21. Downstream Processing Equipment Lab Scale Chromatography System Large Scale Chromatography 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 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

  24. Gel Filtration Chromatography

  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 + 0 -

  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.

  28. Affinity Chromatography Abs 280nm Time (min)

  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

  31. 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

  32. Conductivity Meter UV Detector Injector Valve Column Buffer Select Mixer Peristaltic Pump

  33. The Virtual Chromatography Laboratory http://ATeLearning.com/BioChrom/

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