BIOMAN 2011 CHO-tPA Production System Downstream Processing Mike Fino MiraCosta College

Unit Operations Many decisions to be made at each step in the process

Downstream Example

Harvest Separation (Clarification) There are two technologies for removing the cell mass from the solution containing the target protein prior to loading onto columns: Centrifugation (e.g. disk stack) Filtration Dead-ended filtration (aka normal flow: membrane + depth) Crossflow membrane filtration (aka tangential flow) Crossflow membranes are preferred for large scale operations and have many advantages

Media and Cells In, Clarified Media Out CLARIFIED BROTH SLUDGE 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 a valve.

NORMAL FLOW FILTRATION (NFF): 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.   MEMBRANE DEPTH

Sterilizing Filters: Industry/Regulatory standard Capable of achieving an LRV >7 for a B. diminuta challenge using ASTM methodology (per FDA Guidelines) > 7 LRV means <1 microbe / 107 microbe challenge Doesn’t specify pore size or filter type B. diminuta model organism Sterilizing filters must be able to retain all challenge microorganisms at a maximum bioburden

Tangential Flow Filtration Clarification/Purification Concentration Buffer Exchange Uses Crossflow to reduce build up of retained components on the membrane surface Allows filtration of high fouling streams or high resolution TFF systems often use the same types of filter materials as NFF, but they are operated in at different manner. The feed stream is not pumped directly through the filter, but across the filter, or at a tangent to the filter surface, in order to provide a sweeping action or turbulent flow at the filter surface. Because the feedstream is under pressure, some fluid will still pass through the filter. The goal of TFF is to keep the retained material in the fluid stream and off the filter, to prevent filter plugging. The advantage of TFF is the ability to control fouling allowing the processing of very dirty feedstreams, or the use of membranes with very small pore sizes. The disadvantage of TFF is the requirement for additional process controls and the need to control to fine tolerance.

Different Size Pores in TFF

What is Membrane Integrity? Integral Membrane Non-Integral Membrane Contaminants larger than pores upstream Contaminants larger than expected pores upstream Idealized look at bubble point. On the left the integral membrane pores a filled with liquid which is held there by capillary forces. This liquid and these forces present a barrier to air flow. On the right, the air pressure has exceeded the capillary forces of the largest pore, air flows freely through the pore, and can be seen downstream. Remember that this is an "idealized" model. No downstream contamination Downstream contamination

Principles of Integrity Testing A benefit of membrane filters is the ability to perform a non-destructive integrity test. Testing ensures filtration SYSTEM integrity before, during, or after filtration. Membrane prefilters and depth filters cannot be integrity tested with precision or accuracy because of wide pore distribution.

Reasons to Integrity Test Confirms manufacturers specifications Assures integrity after steaming or autoclaving Assures integrity before sterilization Detects system leaks due to o-rings, gaskets, faulty seals Assures the correct pore size filter Part of corporate standard operating procedure GMP requirement Audit requirement

Two Basic Types of Integrity Test Destructive Provided as a manufacturers assurance of microbial retention. Bacterial Challenge Non-Destructive Provided to allow in-situ testing Pressure hold Bubble Point Diffusion

Basic Elements of a Bacterial Retention Test Saline lactose media w/ B. Diminuta Test Filter 0.22 or 0.1 m disc or filter cartridge Assay Filter (47mm MEC disc) 47mm disc on TSA MEC = mixed esters of cellulose TSA = tryptic soy agar

Non-Destructive Integrity Test Bubble Point Fully wetted membrane filters hold liquid in their pores by surface tension and capillary forces. Bubble point pressure is inversely related to largest pore diameter Open pore space Water held with surface tension View of the membrane cross-section

What is Pressure Hold/Bubble Point? Water Wet Integral Membrane Non-Integral Membrane Air pressure upstream greater than specification Gas will flow through large opening and is easily observed downstream Water in pores is a barrier to gas flow: No gas flow observed downstream until upstream pressure exceeds critical value less than specification psi Idealized look at bubble point. On the left the integral membrane pores a filled with liquid which is held there by capillary forces. This liquid and these forces present a barrier to air flow. On the right, the air pressure has exceeded the capillary forces of the largest pore, air flows freely through the pore, and can be seen downstream. Remember that this is an "idealized" model.

Inverse Relationship: Pore size v. Bubble Point A sterilizing filter has a log reduction value of greater than 7 Decreasing pore size

TFF System

Retentate Flow Permeate Flow Feed Flow Outlet Pressure Hollow Fiber Inlet Pressure

PERISTALTIC PUMP: Creates a gentle squeezing 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 PERISTALTIC PUMP: Creates a gentle squeezing action to move fluid through flexible tubing.

Introduction: TFF Layout & Operation Operating Steps: Flush Clean Water Flux Pump curve Integrity Test Buffer Flush Microfilter Or Concentrate Or Diafilter feed pump filter retentate diafiltrate product recovery initial feed reservoir permeate

Key Parameters Feed Flow rate Transmembrane pressure (TMP) Flow rate leaving the pump Set by pump speed Transmembrane pressure (TMP) Average of inlet/outlet pressures Set by backpressure (retentate) Permeate control Flow rate through the fibers Set by backpressure (permeate) We don’t use this control in this cllass Membrane area Scales linearly

Transmembrane Pressure (TMP) Inlet Feed Pressure Retentate Pressure TMP = (Pin + Pout)/2 - Pperm Pin = 30psi Pout = 20psi Permeate Pressure We leave this line unrestricted Filter membrane Pperm = 0psi TMP = (30 + 20)/2 - 0 = 25 PSI

System Operation Steps Clean water flux Pump Curve Integrity Test Initial Feed Diafiltration Buffer Steps Clean water flux Pump Curve Integrity Test Filtration Flush Retentate Tank Pump Membrane Feed Permeate

Operation: Microfiltration Trash Collect and Keep

Operation: Microfiltration Trash Collect and Keep

Operation: Microfiltration Trash Collect and Keep

Operation: Microfiltration Trash Collect and Keep

Operation: Microfiltration Trash Collect and Keep

Operation: Microfiltration Trash Collect and Keep

Operation: Concentration Diafiltration Buffer Initial Feed Retentate Permeate Feed Tank Pump Membrane Flush Dewater the retained solutes Procedures Fill tank with process fluid Start pump and adjust system to recommended flows/pressures Remove permeate

Operation: Concentration

Operation: Concentration

Operation: Concentration

Operation: Concentration

Operation: Concentration

Operation: Concentration

Operation: Concentration

Operation: Concentration

Operation: Diafiltration “Wash out” permeable solutes- product or contaminants Procedure: Add diafiltration buffer to the feed tank at the same rate that permeate is being removed from the system Diafiltration Buffer Initial Feed Retentate Permeate Feed Tank Pump Membrane Flush

Operation: Diafiltration

Operation: Diafiltration

Operation: Diafiltration

Operation: Diafiltration

Operation: Diafiltration

Operation: Diafiltration

Operation: Diafiltration

Operation: Diafiltration

Background: Virus Safety Effective Clearance Steps Virus Filtration Large (enveloped) & small (non-enveloped) viruses Smallest parvovirus is about 50% bigger than an antibody Inactivation Low pH or Solvent detergent (enveloped) Chromatography Protein A Affinity for MAbs (enveloped & non-enveloped) Anion Exchange Flow through for MAbs (enveloped & non-enveloped)

Types of Chromatography

Column Chromatography

Commonly employed downstream processing methods Attributes Benefits Limitations Clarification: Sedimentation based clarification Continuous centrifugation Capable of handling very large harvest volumes Open process- contamination and safety issues Normal flow Filtration Microporous Volume and throughput limited Charged filter media Cellulose pads Tangential flow filtration Contained systems Capable of handling large harvest volumes Capture: Chromatography Protein A Affinity High throughput, high purity High initial cost Other affinity ligands High throughput Purity, regulatory acceptance Cation exchange Low cost media Low throughput, feedstock preconditioning Purification: Ion exchange, HIC, IMAC, hydroxyapatite Variety of selectivities, high capacity, robust Often flow rate limited Adsorptive membrane Charged membranes High throughput, contained, suited to trace contaminant removal Low capacities

Typical contaminant clearance values from each chromatography stage Affinity load Intermediate purification load Polishing load Host cell protein (ng/ml) 105 103 10 Endotoxin (EU/ml) 106 <1 DNA (pg/ml) 102

Common process constituents and methods of removal or purification 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 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 <0.1-10 mg/l Buffer Growth media Stability media UF Extractables/leachables UF/ Chromatography Purification reagents <0.1-10mg/l

Downstream Design 95% yield/step 90% yield/step 85% yield/step

Ion Exchange Chromatography If the charge on the bead is positive, it will bind negatively charged molecules. This technique is called anion exchange. If the beads are negatively charged, they bind positively charged molecules This technique is called cation exchange.

IEC (cont’d) Thus, a scientist picks the resin to used based on the properties of the protein of interest. During the chromatography, the protein binds to the oppositely charged beads. Once the contaminant protein is separated from the protein of interest, a high salt buffer is used to get the desired protein to elute from the column.

Ion Exchangers Ion exchange chromatography is based on adsorption and reversible binding of charged sample molecules to oppositely charged groups attached to an insoluble resin The pH value at which a biomolecule carries no net charge is called the isoelectric point (pI)

IEX (cont’d) When exposed to a pH below its pI, the biomolecule will carry a positive charge and will bind to a cation exchanger. At a pH above its pI, the protein will carry a negative charge and will to bind to an anion exchanger Depending on what pH the biomolecule is more stable at will decide whether an anion or cation exchanger is used

Background for IEC of tPA SP Sepharose is a cation resin, which means that positively charged molecules will bind to the negatively charged resin. The extent of binding is dependent on the cationic strength of the protein of interest and can be manipulated by changing the pH and/or conductivity of the buffers used in the chromatography process.

The main proteins in the media used to grow tPA are tPA, Bovine serum albumin (BSA), insulin, and transferrin. Each protein has a specific isoelectric point called the pI. BSA has a pI of 4.9 tPA is 7.5 - 8.5 transferrin is 5.9 insulin is 5.3

We are able to selectively bind the tPA to the resin by controlling the pH and ionic strength of the equilibration buffer (aka Buffer A). At a pH of 6.0, tPA is more cationic (positively charged) than either BSA or Transferrin. Therefore, the more positive charged tPA will bind to the resin and the others will flow through the column and out to waste. tPA is then removed from the column using a high concentration of salt, which competitively "bumps" the protein off the resin as the sodium ions bind.

Steps in Chromatography Prime and de-bubble the system Condition the column resin with a solution that promotes the binding of your protein Called Equilibration Pump your sample solution over the column resin, which should bind as much of your protein as possible Called Applying Sample Everything that doesn’t bind goes to the drain. At this point, your protein will stay bound to the resin indefinitely. Now pump a solution over the resin that competes for binding on the resin with the proteins from your solution. Called Elution At some point, the competing solution will beat out the various proteins for position on the resin and they will let go of the resin. You will collect fractions along the way that can be frozen and analyzed later.

ÄktaPrime Liquid Chromotography System

ÄktaPrime Flow Path