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

MSKGEELFTGVVPVLVELDGDVNGQKFSVSGEGEGDATYGKLTLNFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFYKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKMEYNYNSHNVYIMGDKPKNGIKVNFKIRHNIKDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMILLEFVTAARITHGMDELYK

Tertiary Structure

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) =

Tissue Plasminogen Activator

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 favegpklvw

601 stqtala

MW 69 kD pI 5.82 Hydrophobicity -.395

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

Some Other Proteins of Interest

Typical Production Process Flow

(Feed 2)

(Feed 3)

(Feed 4)

Chrom 1 Chrom 3

Cryo-preservation

Concentration /

Diafiltration

Centrifuge

Viral Removal

Filtration

(Feed1) Inoculum Expansion

(Spinner Bottles) Ampule Thaw

Chrom 2

Media Prep

Working Cell Bank

Sub- Culture

Inoculum

Sub- Culture

Sub- Culture

Sub- Culture

Sub- Culture

Large Scale Bioreactor

Wave Bag

Seed Bioreactors

Fermentation

150L Bioreactor

750L

Bioreactor

5,000L

Bioreactor

26,000L

Bioreactor

Depth

Filtration

Collection

Centrifuge

Harvest/Recovery

Harvest

Collection

Tank

1,500L

Filter

Chromatography

Skid

Anion Exchange Chromatography (QXL)

Column Eluate

Hold Tank

8,000L

Eluate

Hold Tank

6,000L

Filter

Chromatography

Skid

Protein A Chromatography

Column

Chromatography

Skid

Column

Eluate

Hold Tank

20,000L

Hydrophobic Interaction Chromatography (HIC)

Eluate

Hold Tank

20,000L

Viral Inactivation

Eluate Hold

Tank

5,000L

Filter

Chromatography

Skid

Anion Exchange Chromatography (QFF - Fast Flow)

Column

Post-viral Hold

Vessel

3,000L

Viral Filtering Ultra Filtration Diafiltration

Bulk

Fill

Purification

24 days 31 days

8 days

1 day

Upstream/Downstream Manufacturing Overview

Clarification or

Removal of Cells and Cell Debris

Using Centrifugation

Using Depth Filtration

Control Panel

Cut-away view

Protective enclosure

Basic components of a centrifuge

Door

Rotor

Drive shaft

Motor

Centrifugal force

Sedimentation path of

particles Pellet deposited at an angle

Center of rotation rminimum

raverage rmaximum

Centrifuge

An instrument that generates centrifugal force. Commonly used to separate particles in a liquid from the liquid.

Continuous Centrifugation

Media and Cells In & Clarified Media Out

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.

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.

Normal Flow Filtration

Depth Filtration:

Equipment

Depth Filtration: Cells and Cellular Debris

Stick to Ceramic Encrusted Fibers in Pads

PROTEIN of INTEREST

Uses crossflow to reduce build up

of retained components on the

membrane surface

Allows filtration of high fouling

streams and high resolution

Tangential Flow Filtration

vs. Normal Flow Filtration

Tangential Flow Filtration

vs. Normal Flow Filtration

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.

How TFF Concentrates and Purifies

a Protein of Interest

Downstream Processing Equipment

Lab-Scale TFF System

Large-Scale TFF System

Low Pressure Production

Chromatography

The System: Components and Processes

The Media: Affinity, Ion Exchange,

Hydrophobic Interaction Chromatography

and Gel Filtration

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

Peristaltic Pump

• Creates a gentle

squeezing action to

move fluid through

flexible tubing.

UV Detector

Detects proteins coming out of the column

by measuring absorbance at 280nm

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)

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

Liquid Column Chromatography

Lonza, Portsmouth, NH

A Commercial LP LC

Chromatography Column

Downstream Processing Equipment

Lab Scale

Chromatography System

Large Scale

Chromatography System

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.

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

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

Gel Filtration Chromatography

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.

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 -

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.

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.

Time (min)

Abs 280nm

Affinity Chromatography

Component Culture Harvest Level

Final Product Level

Conventional Method

Therapeutic Antibody 0.1-1.5 g/l 1-10 g/l UF/Cromatography

Isoforms Various Monomer Chromatography

Serum and host proteins 0.1-3.0 g/l < 0.1-10 mg/l Chromatography

Cell debris and colloids 106/ml None MF

Bacterial pathogens Various <10-6/dose MF

Virus pathogens Various <10-6/dose (12 LRV) virus filtration

DNA 1 mg/l 10 ng/dose Chromatography

Endotoxins Various <0.25 EU/ml Chromatography

Lipids, surfactants 0-1 g/l <0.1-10 mg/l Chromatography

Buffer Growth media Stability media UF

Extractables/leachables Various <0.1-10 mg/l UF/ Chromatography

Purification reagents Various <0.1-10mg/l UF

Common Process Compounds and Methods of Removal or Purification

Biopharmaceutical Production Overview

Typical Process Flow

(Feed 2)

(Feed 3)

(Feed 4)

Chrom 1 Chrom 3

Cryo-preservation

Concentration / Diafiltration

Centrifuge

Viral Removal Filtration

(Feed1) Inoculum Expansion Ampoule Thaw

Chrom 2

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

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).

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

Conductivity Meter

UV Detector

Mixer

Peristaltic Pump

Column

Injector Valve

Buffer Select

Chromatography Skid – Components

Engineering and Advanced Technology

A screenshot of the Virtual Liquid Chromatography Laboratory.

3D images of major system components are delivered as you click on them.

Chromatography Skid – Controller

Engineering and Advanced Technology

The Virtual Liquid Chromatography Laboratory showing the interactive controller which

enables students to operate the system and set process parameters.

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).

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

tR

w1/2

Calculating Column Efficiency (N)

N = 5.54 (tR/w1/2)2

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.

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.

The Virtual Chromatography Laboratory

URL: http://ATeLearning.com/BioChrom/

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