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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 QC Biochemistry. Know the Characteristics of Your Protein Green Fluorescent Protein (GFP). Sequence of Amino Acids.

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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 QC Biochemistry

  2. Know the Characteristics of Your Protein Green Fluorescent Protein (GFP) Sequence of Amino Acids Tertiary Structure MSKGEELFTGVVPVLVELDGDVNGQKFSVSGEGEGDATYGKLTLNFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFYKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKMEYNYNSHNVYIMGDKPKNGIKVNFKIRHNIKDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMILLEFVTAARITHGMDELYK

  3. Know the Characteristics of Your Protein Green Fluorescent Protein (GFP) • MW (molecular weight = 27,000 Daltons (27 kD) • pI (isoelectric point) = 4.8 • Hydropathicity (=hydrophobicity) =

  4. Some Other Proteins of Interest Tissue Plasminogen Activator 1 rrgarsyqvi crdektqmiy qqhqswlrpv lrsnrveycw cnsgraqchs vpvkscsepr 61 cfnggtcqqa lyfsdfvcqc pegfagkcce idtratcyed qgisyrgtws taesgaectn 121 wnssalaqkp ysgrrpdair lglgnhnycr npdrdskpwc yvfkagkyss efcstpacse 181 gnsdcyfgng sayrgthslt esgasclpwn smiligkvyt aqnpsaqalg lgkhnycrnp 241 dgdakpwchv lknrrltwey cdvpscstcg lrqysqpqfr ikgglfadia shpwqaaifa 301 khrrspgerf lcggilissc wilsaahcfq erfpphhltv ilgrtyrvvp geeeqkfeve 361 kyivhkefdd dtydndiall qlksdssrca qessvvrtvc lppadlqlpd wtecelsgyg 421 khealspfys erlkeahvrl ypssrctsqh llnrtvtdnm lcagdtrsgg pqanlhdacq 481 gdsggplvcl ndgrmtlvgi iswglgcgqk dvpgvytkvt nyldwirdnm rp MW 60 kD pI: 8.04 Hydrophobicity -.516 Human Serum Albumin mkwvtfisll llfssaysrg vfrrdthkse iahrfkdlge ehfkglvlia fsqylqqcpf 61 dehvklvnel tefaktcvad eshagceksl htlfgdelck vaslretygd madccekqep 121 ernecflshk ddspdlpklk pdpntlcdef kadekkfwgk ylyeiarrhp yfyapellyy 181 ankyngvfqe ccqaedkgac llpkietmre kvltssarqr lrcasiqkfg eralkawsva 241 rlsqkfpkae fvevtklvtd ltkvhkecch gdllecaddr adlakyicdn qdtissklke 301 ccdkplleks hciaevekda ipenlpplta dfaedkdvck nyqeakdafl gsflyeysrr 361 hpeyavsvll rlakeyeatl eeccakddph acystvfdkl khlvdepqnl ikqncdqfek 421 lgeygfqnal ivrytrkvpq vstptlvevs rslgkvgtrc ctkpesermp ctedylslil 481 nrlcvlhekt pvsekvtkcc teslvnrrpc fsaltpdety vpkafdeklf tfhadictlp 541 dtekqikkqt alvellkhkp kateeqlktv menfvafvdk ccaaddkeac favegpklvw601 stqtala MW 69 kD pI 5.82 Hydrophobicity -.395

  5. 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

  6. Upstream/Downstream Manufacturing 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

  7. Clarification or Removal of Cells and Cell Debris Using Centrifugation Using Depth Filtration

  8. rminimum Centrifugal force Sedimentation path of particles Center of rotation Control Panel raverage Pellet deposited at an angle rmaximum Protective enclosure Door Cut-away view Rotor Drive shaft Motor Basic components of a centrifuge Centrifuge An instrument that generates centrifugal force. Commonly used to separate particles in a liquid from the liquid.

  9. Continuous Centrifugation Media and Cells In & Clarified Media Out

  10. 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.

  11. Normal Flow Filtration Traps contaminants larger than the pore size on the top surface of the membrane. Contaminants smaller than the specified pore size pass through the membrane. Used for critical applications such as sterilizing and final filtration.  

  12. Depth Filtration: Equipment

  13. Depth Filtration: Cells and Cellular Debris Stick to Ceramic Encrusted Fibers in Pads PROTEIN of INTEREST

  14. Tangential Flow Filtration vs. Normal Flow Filtration Uses crossflow to reduce build up of retained components on the membrane surface Allows filtration of high fouling streams and high resolution

  15. Tangential Flow Filtration vs. Normal Flow Filtration

  16. 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.

  17. How TFF Concentrates and Purifiesa Protein of Interest

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

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

  20. 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

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

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

  23. 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)

  24. 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

  25. Liquid Column Chromatography

  26. A Commercial LP LC Chromatography Column Lonza, Portsmouth, NH

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

  28. 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.

  29. Liquid Chromatography Protein solution is applied to a column Column filled with matrix (stationary phase) + liquid phase (mobile phase)‏ Proteins separated based on differing affinity for the stationary and mobile phases 1 2 3 4

  30. 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

  31. Gel Filtration Chromatography

  32. 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.

  33. 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 -

  34. Hydrophobic Interaction Chromatography (HIC) HIC is finding dramatically increased use in production chromatography. Since the molecular mechanism of HIC relies on unique structural features, it serves as an orthogonal method to ion exchange and affinity 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. These special properties make HIC very useful in whole processes for bridging or transitioning between other steps in addition to the separation which is effected.

  35. Affinity Chromatography Affinity chromatography separates proteins on the basis of a reversible interaction between a protein and a specific ligand coupled to a chromatography matrix. With high selectivity, hence high resolution, and high capacity for the protein(s) of interest, purification levels in the order of several thousand-fold with high recovery of active material are achievable. Target protein(s) is collected in a purified, concentrated form. Biological interactions between ligand and target molecule can be a result of electrostatic or hydrophobic interactions, van der Waals' forces and/or hydrogen bonding. To elute the target molecule from the affinity medium the interaction can be reversed, either specifically using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. In a single step, affinity purification can offer immense time-saving over less selective multistep procedures. The concentrating effect enables large volumes to be processed. Target molecules can be purified from complex biological mixtures, native forms can be separated from denatured forms of the same substance and small amounts of biological material can be purified from high levels of contaminating substances.

  36. Affinity Chromatography Abs 280nm Time (min)

  37. Common Process Compounds and Methods of Removal or Purification

  38. Concentration / Diafiltration (Feed 2) (Feed1) Centrifuge Chrom 1 Chrom 2 Cryo-preservation (Feed 4) Biopharmaceutical Production Overview Typical Process Flow Inoculum Expansion Ampoule Thaw (Feed 3) Chrom 3 Viral Removal Filtration

  39. What Will Change During Scale-up?Process Development Considerations • Utility requirements • Water requirement • Cleaning/Sanitizing solution requirements • Buffer prep • Number of steps in cell culture scale up • Harvest techniques • Column packing; distribution of introduced liquid at large columns • Equipment – bubble trap • Automation of process • Data collection • Sample load

  40. Virtual Chromatography – The Power of Interactive Visualization in Understanding a STEM Field of Study • Understanding the physics, chemistry and biology of the chromatographic system and the binding of the protein of interest to the chromatographic matrix or beads (Science) • Understanding the design and operation of chromatography components and of the chromatographic process (Technology and Engineering). • Understanding the calculations needed to run the chromatographic system (column volume) and the measurements on chromatograms needed to calculate the HETP, number of theoretical plates, retention time, and resolution (Mathematics).

  41. 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

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

  43. Chromatography Skid – ComponentsEngineering and Advanced Technology A screenshot of the Virtual Liquid Chromatography Laboratory. 3D images of major system components are delivered as you click on them.

  44. Chromatography Skid – ControllerEngineering and Advanced Technology The Virtual Liquid Chromatography Laboratory showing the interactive controller which enables students to operate the system and set process parameters.

  45. Chromatography Skid – Chromatogram with Mathematics The Virtual Chromatography Laboratory teaches students how to make calculations on chromatograms such as the efficiency of column packing (HETP).

  46. Height Equivalent to Theoretical Plate (HETP) • The smaller the HETP the better • Allows comparison of columns of different lengths • Column length expressed in mm HETP = L/N

  47. w1/2 tR Calculating Column Efficiency (N) N = 5.54 (tR/w1/2)2

  48. Chromatography Skid – Chromatography Science and Technology The Virtual Chromatography Laboratory showing the operation of the chromatography system during the ‘load’ phase, the chromatogram showing the flow through of proteins that do not attach to the chromatographic matrix, and a nanoscale view inside the column of the affinity bead with the protein of interest in the filtrate (green) attached and proteins not specific for the bead flowing through the column.

  49. Chromatography Skid –Chromatography Science and Technology The Virtual Chromatography Laboratory showing the operation of the chromatography system during the ‘elution’ phase, the chromatogram showing the beginning of the peak of the protein of interest, and a nanoscale view inside the column of the affinity bead showing the protein of interest detaching from the bead as the elution buffer (red) moves through the column.

  50. The Virtual Chromatography Laboratory URL: http://ATeLearning.com/BioChrom/ To login enter your email address and the password: teachbio

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