Downstream Processing in Biopharmaceutical Manufacturing

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

2. Biotherapeutics • Produced in genetically engineered host cells • More than 70% of therapeutic proteins produced in CHO cell culture • Lead products include proteins, vaccines, MAbs • Complex, heterogeneous • 3D Structure • Post-translational modifications • Slight process changes affect activity • Highly regulated processes • Process is the product B. Leader et al., Nat. Rev. Drug Discov 2008, 7:21-39

3. Market for Biopharmaceuticals • 275 biotherapeutics and vaccines approved by the FDA • 425 additional drugs and vaccines for more than 100 diseases in various phases of clinical trials • Overall biologics market: $135 billion in 2009, $239 billion by 2015 (> 9% growth rate) • Protein therapeutics market: $95 billion in 2008, $160 billion in 2013 • Major components of biopharmaceutical market in 2009: • MAbs, $38 billion, 15% CARG • Proteins, $61 billion • Vaccines, $18 billion Univ. of Birmingham College of Medical and Dental Sciences The Future of Biologics Market, Business Insight, March 2009, p. 19 Biologic Therapeutic Drugs: Tech. and Global Markets, BCC Research, Jan. 2011

4. Annual Production of Selected MAbs[2009, GE Healthcare] High value products ~ Several million $ per kg

5. Biologics in FDA Pipeline[GE Healthcare, from 2008 PhRMA Report] • Antisense 16 • Cell therapy 23 • Gene therapy 38 • Growth factors 5 • Interferons 16 • Interleukins 10 • Monoclonal antibodies (mAbs) 192 • Recombinant proteins 66 • Vaccines 223 • Others 44 Over 400 protein products awaiting FDA approval

6. 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

7. 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

8. 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

9. 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

10. Clarification or Removal of Cells and Cell Debris Using Centrifugation (Using Depth Filtration)

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

12. 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.

13. 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

14. 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.

15. 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

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

17. How TFF Concentrates and Diafiltersthe Protein of Interest

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

19. 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

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

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

22. 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)

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

25. Protein Purification

26. 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.

27. Column chromatography • After the initial fractionation steps we move to column chromatography. • The mixture of substances (proteins) to be fractionated is dissolved in a liquid or gaseous fluid called the mobile phase. • This solution is passed through a column consisting of a porous solid matrix called the stationary phase. These are sometimes called resins when used in liquid chromatography. • The stationary phase has certain physical and chemical characteristics that allow it to interact in various ways with different proteins. • Common types of chromatographic stationary phases • Ion exchange • Hydrophobic • Gel filtration • Affinity

28. 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

29. Gel Filtration Chromatography

30. Gel filtration chromatography • Also called size exclusion chromatography or molecular sieve chromatography. How does it work? If we assume proteins are spherical… Molecular mass (daltons) 10,000 30,000 100,000 size

31. Gel filtration chromatography flow

32. Gel filtration chromatography flow

33. Gel filtration chromatography flow

34. Gel filtration chromatography flow

35. Gel filtration chromatography flow

36. Gel filtration chromatography • The molecular mass of the smallest molecule unable to penetrate the pores of the gel is at the exclusion limit. • The exclusion limit is a function of molecular shape, since elongated molecules are less likely to penetrate a gel pore than other shapes. • Behavior of the molecule on the gel can be quantitatively characterized. Total bed volume of the column Vt = Vx + V0 Vx = volume occupied by gel beads V0 = volume of solvent space surrounding gel; Typically 35%

37. 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 -

38. 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.

39. Negatively charged (acidic) protein or enzyme - - - - Ion exchange chromatography • Ion exchange resins contain charged groups. • If these groups are acidic in nature they interact with positively charged proteins and are called cation exchangers. • If these groups are basic in nature, they interact with negatively charged molecules and are called anionexchangers. Positively charged (basic) protein or enzyme + CH2-COO- + CH2-COO- + + CM cellulose cation exchanger CH2-CH2 -NH+(CH2CH2) CH2-CH2 -NH+(CH2CH2) DEAE cellulose anion exchanger

40. CH2-COO- CH2-COO- CM cellulose cation exchanger Negatively charged proteins pass through the column - - - - - - - - Ion exchange chromatography For protein binding, the pH is fixed (usually near neutral) under low salt conditions. Example cation exchange column… Positively charged protein or enzyme bind to the column + + + +

41. CH2-COO- CH2-COO- CH2-COO- CH2-COO- + + CM cellulose cation exchanger CM cellulose cation exchanger + + + + + + Ion exchange chromatography To elute our protein of interest, add increasingly higher amount of salt (increase the ionic strength). Na+ will interact with the cation resin and Cl- will interact with our positively charged protein to elute off the column. + Increasing [NaCl] of the elution buffer Cl- Na+ Na+2 Cl- Na+ Cl- Cl- Na+2

42. Ion exchange chromatography • Proteins will bind to an ion exchanger with different affinities. • As the column is washed with buffer, those proteins relatively low affinities for the ion exchange resin will move through the column faster than the proteins that bind to the column. • The greater the binding affinity of a protein for the ion exchange column, the more it will be slowed in eluting off the column. • Proteins can be eluted by changing the elution buffer to one with a higher salt concentration and/or a different pH (stepwise elution or gradient elution). • Cation exchangers bind to proteins with positive charges. • Anion exchangers bind to proteins with negative charges.

43. Ion exchange chromatography using stepwise elution. Page 134

44. Ion exchange chromatography • Gradient elution can improve the washing of ion exchange columns. • The salt concentration and/or pH is continuously varied as the column is eluted so as to release sequentially the proteins bound to the column. • The most widely used gradient is the linear gradient where the concentration of eluant solution varies linearly with the volume of the solution passed. • The solute concentration, c, is expressed as c = c2 - (c2 - c1)f c1= the initial concentration of the solution in the mixing chamber c2= the concentration of the reservoir chamber f= the remaining fraction of the combined volumes of the solutions initially present in both reservoirs.

45. Figure 6-7 Device for generating a linear concentration gradient. Page 135 c = c2 - (c2 - c1)f

46. 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.

47. Affinity Chromatography Abs 280nm Time (min)

48. 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.

49. Hexamer Peptide Ligands that Mimic Protein A Fab F(ab’)2 Fc Fc used as a fusion tag Structure of IgG Univ. of Birmingham College of Medical and Dental Sciences  InvivoGen.http://www.invivogen.com

50. Hexamer Peptides Selective for Fc Fragment HWRGWV, HYFKFD andHFRRHLpeptide ligands identified from the screening procedure were used for chromatographic binding study of IgG fragments Fc fragment binding Fab fragment binding Elution pH 4 Regeneration Elution pH 4 Load Regeneration Load Peptide ligands bind to the Fc region of IgG, similar to Protein A