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) =

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

5. LSCC Mfg Process Overview
Large Scale Bioreactor
Wave
Bag
Seed Bioreactors
Fermentation
150L Bioreactor
750L Bioreactor
5,000L Bioreactor
26,000L Bioreactor
1 day
Media Prep
Working Cell
Bank
Sub-
Culture
Inoculum
Depth
Filtration
Collection
Centrifuge
Harvest/Recovery
24 days
31 days
Harvest
Collection
Tank
1,500L
Filter
Chromatography Skid
Anion Exchange Chromatography (QXL)
Column
Eluate
Hold
8,000L
6,000L
Protein A Chromatography
20,000L
Hydrophobic Interaction Chromatography (HIC)
Viral Inactivation
5,000L
Anion Exchange Chromatography (QFF - Fast Flow)
Post-viral
Vessel
3,000L
Viral Filtering
Ultra Filtration
Diafiltration
Bulk Fill
Purification
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
Continuous multichamber disc-stack centrifuge. The bowl contains a
number 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 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 – 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 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 Diafilters the 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.
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 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 23

24. Gel Filtration Chromatography24

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.

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
Component
Culture Harvest Level
Final Product Level
Conventional Method
Therapeutic Antibody
g/l
1-10 g/l
UF/Cromatography
Isoforms
Various
Monomer
Chromatography
Serum and host proteins
g/l
< mg/l
Cell debris and colloids
106/ml
None MF
Bacterial pathogens
<10-6/dose
Virus pathogens
<10-6/dose (12 LRV)
virus filtration
DNA
1 mg/l
10 ng/dose
Endotoxins
<0.25 EU/ml
Lipids, surfactants
0-1 g/l
< mg/l
Buffer
Growth media
Stability media UF
Extractables/leachables
UF/ Chromatography
Purification reagents
<0.1-10mg/l

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