Downstream Processing in Biopharmaceutical Manufacturing

Harvest and ClarificationTangential Flow Filtration (UF/DF)

Low Pressure Liquid Column Chromatography

Know the Characteristics of Your Protein Human Serum Albumin

Sequence of Amino AcidsMKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGEEHFKGLVLIA FSQYLQQCPFDEHVKLVNEL TEFAKTCVADESHAGCEKSL HTLFGDELCK VASLRETYGMADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEFKADEKKFWGKYLYEIARRHP YFYAPELLYYANKYNGVFQE CCQAEDKGACLLPKIETMRE KVLTSSARQR LRCASIQKFG ERALKAWSVA RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDRADLAKYICDN QDTISSKLKECCDKPLLEKS HCIAEVEKDAIPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKLKHLVDEPQNL IKQNCDQFEKLGEYGFQNAL IVRYTRKVPQVSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETYVPKAFDEKLF TFHADICTLPDTEKQIKKQT ALVELLKHKPKATEEQLKTV MENFVAFVDK CCAADDKEACFAVEGPKLVWSTQTALA

Tertiary Structure

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

Typical Production Process Flow

(Feed 2)

(Feed 3)

(Feed 4)

Chrom 1Chrom 3

Cryo-preservation

Concentration / Diafiltration

Centrifuge

Viral Removal Filtration

(Feed1)Inoculum Expansion(Spinner Bottles)Ampule Thaw

Chrom 2

Media PrepMedia Prep

Working Cell Bank

Working Cell Bank

Sub- Culture

Sub- Culture

Inoculum

Sub- Culture

Sub- Culture Sub-

Culture

Sub- Culture Sub-

Culture

Sub- Culture Sub-

Culture

Sub- Culture

Large Scale Bioreactor

Wave Bag

Wave Bag

Seed Bioreactors

Fermentation

150L Bioreactor

750L Bioreactor

5,000L Bioreactor

26,000L Bioreactor

Depth Filtration

Depth Filtration

CollectionCollection

CentrifugeCentrifuge

Harvest/Recovery

HarvestCollection

Tank

1,500L

HarvestCollection

Tank

1,500L

FilterChromatography

Skid

Anion Exchange Chromatography (QXL)

ColumnEluateHold Tank

8,000L

EluateHold Tank

8,000L

EluateHold Tank

6,000L

EluateHold Tank

6,000L

FilterChromatography

Skid

Protein A Chromatography

Column

Chromatography Skid

Column

EluateHold Tank

20,000L

EluateHold Tank

20,000L

Hydrophobic Interaction Chromatography (HIC)

EluateHold Tank

20,000L

EluateHold Tank

20,000L

Viral Inactivation

EluateHold Tank

5,000L

EluateHold Tank

5,000L

FilterChromatography

Skid

Anion Exchange Chromatography

(QFF - Fast Flow)

Column

Post-viralHold

Vessel3,000L

Post-viralHold

Vessel3,000L

Viral Filtering Ultra FiltrationDiafiltration

Bulk Fill

Purification

24 days 31 days

8 days

1 dayLSCC Mfg Process Overview

Clarification or Removal of Cells and Cell Debris

Using Centrifugation(Using Depth Filtration)

Continuous CentrifugationMedia 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 thefilter are retained by the filter.

Particles smaller than the pore size of the filter pass through the filter along with the liquid.

Filtration

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

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.

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/minFlush retentate lineFlush 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

Downstream Processing Equipment

Lab-Scale TFF System Large-Scale TFF System

Lab-Scale TFF Filter = Pall’s Pellicon

How TFF Concentrates and Diafiltersthe Protein of Interest

Low Pressure Production Chromatography

The System: Components and ProcessesThe 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 ProcessPurge Air from System with Equilibration BufferPack Column with Beads (e.g. ion exchange,

HIC, affinity or gel filtration beads)Equilibrate Column with Equilibration BufferLoad Column with Filtrate containing Protein of

Interest in Equilibration Buffer Wash Column with Equilibration BufferElute Protein of Interest with Elution Buffer of

High or Low Salt or pHRegenerate Column or Clean and Store

Downstream Processing EquipmentLab 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.

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 IEFOnce 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 -

Affinity ChromatographyAffinity 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.

Time (min)

Abs 280nm

Affinity Chromatography

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.

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 Purification or Removal

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

PeristalticPump

Column

Injector Valve

Buffer Select

The Virtual Chromatography Laboratory

http://ATeLearning.com/BioChrom/