Downstream Processing in Biopharmaceutical Manufacturing

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

• MSKGEELFTGVVPVLVELDGDVNGQKFSVSGEGEGDATYGKLTLNFICTT
  GKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFY
  KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKMEYNYNSHNV
  YIMGDKPKNGIKVNFKIRHNIKDGSVQLADHYQQNTPIGDGPVLLPDNHY
  LSTQSALSKDPNEKRDHMILLEFVTAARITHGMDELYK

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
Typical Production Process Flow
Inoculum Expansion (Spinner Bottles)
Ampule Thaw
(Feed 3)
Chrom 3
Viral Removal Filtration
6
Upstream/Downstream Manufacturing Overview
1 day
24 days
31 days
8 days
7
Clarification or Removal of Cells and Cell Debris

• Using Centrifugation
• Using Depth Filtration

8
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/hydroph
  ilic 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 lt pI lt 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
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)
HETP L/N

• The smaller the HETP the better
• Allows comparison of columns of different lengths
• Column length expressed in mm

47
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/

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