Cell Harvesting 2021 Presented by Beth Zielinski-Habershaw, PhD
9:00am-9:30am
INTRODUCTIONS
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What will this this course provide? •
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This course will provide a comprehensive overview of cell harvest operations in biopharmaceutical manufacturing We begin with a basic overview of the theory and practices of cell harvesting. We then continue our discussion of key process goals that have guided current cell harvest methodologies. The last three modules are broken down into the standard cell harvesting processes used by industry
After this course you will •
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You will understand the “whys” behind cell harvest processes including development and applications. You will gain an understanding of the theories behind and methodologies of three basic cell harvesting techniques; precipitation/flocculation, centrifugation and filtration. You will understand concepts and applications for the three cell harvesting processes and thus gain comprehensive training on current industry-based cell harvesting processes and standards.
Table of contents • • •
Cell harvest overview Goals of the cell harvest process Processes used by industry • •
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Precipitation Centrifugation Filtration
9:30am-11:00am
CELL HARVEST OVERVIEW & DEVELOPMENT
2
Cell Harvest Overview
Cell harvest options
Harvest clarification options; Bioprocess International
Centrifuge Bioreactor
Harvest Centrate Surge Tank
Depth Filtration
Membrane Filtration Harvest Filtrate Hold Tank
Figure 2-1: Harvest Flow Diagram
Courtesy of Amgen
What is cell harvesting? •
Removal of cells, fine particulates, colloids, and soluble impurities prior to the initial purification Centrifuge steps (chromatography) Bioreactor
Harvest Centrate Surge Tank
Depth Filtration
Membrane Filtration Harvest Filtrate Hold Tank
Figure 2-1: Harvest Flow Diagram
Courtesy of Amgen
A bit of history………..
Why separate cells from protein products, cellular waste and debris? Therapeutic protein products are either secreted from cells or stored in intracellular vesicles. Therapeutic proteins must be isolated from ALL other cellular components, debris and waste products/impurities and this occurs during cell harvest and purification steps. Prior to purification (chromatography), cell separation methods provide a clean stream of drug substance that can be introduced to subsequent chromatography steps. Cell harvesting methods reduce the chances of phase fouling during purifying chromatography steps.
What are we separating? 1
Therapeutic protein bound in vesicles
2
Secreted therapeutic protein
Open question: What species could the cells above these represent?
What are the primary goals of cell separation? Reproducible or consistent removal of cells and debris High product yield (therapeutic antibody concentration) Reduction of impurities We will discuss these terms in a few slides!
More efficient engineering of CHO cells and more effective means of cell culture during upstream processing (fed batch) has led to cell batches that contain up to 50 million cells per ml and produce greater than 10 grams per liter of therapeutic protein!
By Leebatharushon, [1] - Own work, CC BY-SA 4.0,
Courtesy: Rick Lawless
Fed batch production can yield > 10g/L from 50 million cells/ml! Fed batch
Solid separation Cells, debris, insoluble media components
Solid impurity removal
Continuous processing
HCP-Host cell protein DNA-Deoxyribonucleic acid HMW-High molecular weight LMW-Low molecular weight
Regardless of the mode of cell culture, the therapeutic proteins must be separated from the rest of the contents! Open question: What is continuous production?
Batch, fed batch and continuous cultures Batch: No extra feeding is used from beginning to end of the process
Fed-batch: Feeding with substrate and supplements can extend the duration of culture for higher cell densities or switch metabolism to produce e.g. a recombinant protein
Continuous culture: Either feed rate of a growth-limiting substance keeps cell density constant (a chemostat) or cell density determines the feed rate of the substrate (turbidostat). Cell retention/perfusion. Incoming feed rate matches the rate of removal of harvest. Steady state can be achieved which can last for days to months. Long-term production
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General methods for separating cells from protein products, cellular waste, and debris Intracellular products (ex. E.Coli derived protein products) Cell disruption Physical Enzymatic
Extracellular products (ex. Most CHO cell derived products) Precipitation/flocculation Centrifugation Filtration Depth filtration Tangential flow microfiltration
Courtesy of Amgen
What is process yield and retention? Remember our goal: To clarify the cell suspension such that cells, debris and other particulate waste are separated from the therapeutic protein prior to downstream purification steps. Yield: Amount (g/L) of substance of interest collected following harvest processing steps
Retention: Amount (g/L) of substance left behind following harvest processing steps
Harvesting for batch processing
Amgen’s single use systems use flocculation then depth filtration Boehringer Ingelheim 2017
Harvesting for continuous processing
ISPE.org
Substrate
pO2
https://upload.wikimedia.org/wikipedia/commons/thumb/c/cc/Fed_batch_principle.png/440pxFed_batch_principle.png
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Challenges for cell culture separation/harvest Cell culture process Product titer DNA/HCP impurities Biomass concentration Cell culture viability
Cell culture media Hydrolysates, peptones Trace elements Buffer system Amino acids High glucose
Questions to consider Why is cell harvest and separation important for your colleagues performing downstream chromatography? A. Removal of bacterial waste only B. Clarification of feed stream of drug substance for optimal downstream processing C. Viral elimination only
Questions to consider Why is separation/purification important for patients who ultimately use a protein therapy? A. Risk mitigation of potential immunological responses to extraneous cellular components and debris B. To provide patients with intended biological theraoeutic only C. Both A & B
Questions to consider Which type of cell requires the least complicated separation process? A. Attachment dependent B. Single cell suspension C. Cell aggregate suspension
Questions to consider What are the primary goals of cell separation? A. Reproducible or consistent removal of cells and debris B. Reduction of impurities C. Both A & B
Questions to consider When harvesting cells that produce a very high protein yield, _______ is the most efficient method for cell harvest A. Membrane filtration B. Centrifugation C. Cellular lysis
Questions to consider What are two major challenges facing cell harvest operations? A. Product titer and cell viability B. Presence of lead and the presence of mercury C. High carbon dioxide and nitrogen levels
Development of the Process
What are the requirements for effective and efficient clarification steps during cell harvest? • Scurran15
Process should be rapid enough to prevent degradation of therapeutic protein products and reduce chances of contamination • How can proteins degrade? • Excessive heat and acid precipitation/flocculation steps can lead to unfolding of proteins • Reduce chances for breaches in sterility • Areas to be aware of: • Connections between the bioreactor and centrifuge/transfer • Addition of exogenous reagents • Collection vessels • Any and all steps that require manipulation of the cell suspension
What are the requirements for effective and efficient clarification steps during cell harvest? Solids must be removed Concentration of solids is critical and can vary between cell lines, cell batches and even media used Typically, centrifugation is best for condensing solids Continuous centrifugation allows for continual discharge of solid waste
Depth and microfiltration (TFF) units are prone to protein fouling on membranes (clogging) This can result in failure of the membrane devices and/or loss of product Larger membrane areas may be helpful but are also more costly
What are the requirements for effective and efficient clarification steps during cell harvest? Consistency between batches of cells in order to reduce variability in downstream (ultimately final) product High yields of post harvest therapeutic proteins Reducing the burden on purification steps downstream
What factors impact recovery of therapeutic protein during the cell harvest process? Cell density: Increased cell density = Increased solid mass (particulates)/volume Impurities may also increase as cells are lysed or ruptured during centrifugation and more DNA and intracellular proteins (not therapeutic target) are released Depth and microfiltration alone is not sufficient when dealing with high cell densities due to high concentrations of solid mass or particulates Zhaopeng Ma, Zhangxi Hu, Yunyan Deng, Lixia Shang, Christophere J. Gobler, Ying Zhong Tang
What factors impact recovery of therapeutic protein during the cell harvest process? Soluble impurities such as some host cell proteins and DNA may remain in the clarified yield. WHO-(10ng/dose)
In addition to further clarification using depth and microfiltration systems, flocculation early in the process may reduce the amount of these contaminants streaming into downstream purification steps DNA extraction from cow thymus Alyssa LaGrange
What factors impact recovery of therapeutic protein during the cell harvest process? Cell viability: Extension of cell culture times to increase protein production can lead to decreases in cell viability This can lead to an increase in the number of particulates released into the cell media Particulate size distribution can impact clarification steps. Centrifugation typically separates most particulates from suspension Density Choice of membranes during microfiltration steps depends upon particulate size, charge and hydrophilicity/phobicity Nominal molecular weight cut off Cells may also be more sensitive to shear forces during centrifugation thus rupturing and releasing cellular content that does not settle into the final waste “pellet”
IsabelMaestre
Particle size and flocculation/ precipitation Particle size is a major determinant of cell separation methodology
Extracellular particle diameter
CHO cell diameter
Bimodal distribution
# of particles/ml
Average particle diameter in µm
Narrow the particle size distribution and your process will be much easier
Questions to consider What are two requirements for effective and efficient clarification steps during cell harvest? A. B. C.
Prevention of protein unfolding Prevention of contamination Prevention of cell lysis
Questions to consider Name one principal factor that impacts the recovery of therapeutic protein during cell harvesting operations? A.
Cell density
B. C.
Concentration of insoluble impurities Neither A nor B
Questions to consider Extracellular particles present in cell culture are typically smaller and less dense than viable cells? A.
True
B.
False
11:00am-11:20am
BREAK – 20 MINUTES
2
11:20am-12:50pm
PRECIPITATION & FLOCCULATION
Courtesy of Amgen
Courtesy of Amgen 45
Theory supporting precipitation methods
Effects of acid on milk
The theory of precipitation is based on the concept of converting soluble components of a solution or suspension (cell suspension) into insoluble particles that separate from the aqueous (water) phase of the solution of suspension
Open question: Is this precipitation or flocculation? Leesean
Acid precipitation
The difference between flocculation and precipitation Both serve the purpose of assisting in the removal of contaminants from a suspension •
Flocculation • Process in which colloidal components equally dispersed in an aqueous suspension can aggregate to form flakes or “flocs”
•
Precipitation • Formation of an insoluble solid mass from a liquid solution; precipitate. • Precipitate is formed when two soluble ionic compounds are mixed.
Precipitation is strongly influenced by…. Temperature pH Ionic strength Protein concentration Protein surface characteristics Distribution of polar and non-polar amino acids Precipitating agent
Developmental methods that have been used for precipitation Precipitation methods are used to separate out either the therapeutic protein or impurities Caprylic acid (impurity precipitation) CaCl2 (impurity precipitation) PEG (mAb product precipitation) Cold ethanol (mAb product precipitation)
Utilizing the methods stated above, studies have shown: mAb yield of 70%, removal of HCPs as low as 300 ppm, removal of DNA up to94 ng/ml (Sommer, R. et al. (2015) Affinity ligands or peptide tags can be used to precipitate therapeutic proteins; complexes form under specific conditions such as changes in pH: studies have shown >85% yield and >95% purity of bivalent active antibodies. (Trends in Biotechnology, March 2019, Vol. 37, No. 3)
Types of precipitation: Acid/Base precipitation pH change
All proteins have an isoelectronic point or pI value (net charge=0) Add acid=positive charge on outside of proteins=repulsive forces between proteins Add base=negative charge on outside of proteins=repulsive forces between proteins pI of most proteins is in the pH range of 4–6 at which point the positive and negative charges cancel each other out and the proteins aggregate If the pH of the cell culture media is approximately 7.0, then acid will have to be added to reach the protein of interest’s pI value at which point protein aggregation will occur
TCA (Trichloroacetic Acid), is commonly used because it is highly reactive and can be used in small amounts. https://glossary.periodni.com/glossary.php?en=isoelectric+point
The proteins remain nonfunctional once structural changes have occurred
Types of precipitation: Precipitation with PEI (polyethylenimine) Sodium Chloride The binding of proteins to PEI is saltdependent. PEI is weakly cationic (+)
PEI-Protein Precipitate (1) PEI (+)
Precipitated proteins are recovered from PEI-protein complexes by elution with NaCl.
Ammonium sulfate precipitation is often used after PEI precipitation and elution, because protein will be precipitated by ammonium sulfate, but the PEI will remain in solution.
Ammonium Sulfate Flocculation (3)
PEI (+)
Elution (2)
PEI (+)
PEI (+) PEI (+) PEI (+)
PEI (+)
Types of precipitation: Alcohol precipitation Alcohol reduces the hydration of the protein, by removing water and surrounding proteins which leads to aggregation and precipitation. DNA and RNA can be precipitated using ethanol To preserve their biological functions, it is important to run the experiments at low temperature. Also, check the pH of the solution and the concentration of the protein. The most commonly used solvents are ethanol, methanol and acetone. Open question: Why is DNA/RNA extracted at low temperatures?
Precipitation for batch processes
Processes 2021, 9(3), 488; https://doi.org/10.3390/pr9030488
Precipitation for perfusion/continuous processing
Process Biochemistry Volume 51, Issue 10, October 2016, Pages 1610-1621
Process Parameters
Courtesy of Amgen
Performance Indicators
Courtesy of Amgen
Precipitates
Figure 4. Light microscopy images of precipitates. (a) shows a magnification of 10-fold and (b) of 50-fold. Processes 2021, 9(3), 488; https://doi.org/10.3390/pr9030488
Flocculation
The difference between flocculation and precipitation Both serve the purpose of assisting in the removal of contaminants from a suspension •
Flocculation • Process in which colloidal components equally dispersed in an aqueous suspension can aggregate to form flakes or “flocs”
•
Precipitation • Formation of an insoluble solid mass from a liquid solution; precipitate. • Precipitate is formed when two soluble ionic compounds are mixed.
Flocculation is strongly influenced by…. Temperature pH Ionic strength Protein concentration Protein surface characteristics Distribution of polar and non-polar amino acids Precipitating agent
Types of flocculation: Ammonium sulfate flocculation following precipitation with PEI (polyethylenimine) Sodium Chloride The binding of proteins to PEI is saltdependent. PEI is weakly cationic (+) Precipitated proteins are recovered from PEI-protein complexes by elution with NaCl. Ammonium sulfate flocculation is often used after PEI precipitation and elution, because protein will be flocculated by ammonium sulfate, but the PEI will remain in solution.
PEI-Protein Precipitate (1) PEI (+) PEI (+)
Elution (2)
PEI (+)
PEI (+) PEI (+)
Ammonium Sulfate Flocculation (3)
PEI (+)
PEI (+)
Types of flocculation: Salting Adding the salt ions into the solution causes restriction of the available water molecules for the proteins. Leads to destruction of the hydrogen bonds. The interactions between proteins is stronger than between the protein and the available water molecules which cause protein aggregation and precipitation. Some examples are zinc sulphate and ammonium sulphate. Not destructive to the protein because aggregates/flocs of proteins form and individual protein structure is not disturbed.
Ammonium salt flocculation Ammonium sulfate is an inorganic salt (no carbon) Ammonium sulfate salt disassociates into ammonium (NH4+) and sulfate (SO42−) in water-based solutions. Ammonium sulfate is highly soluble, stabilizes protein structure, has a low density, inexpensive.
Ammonium salt flocculation
Methods in Enzymology Volume 541, 2014
Solubility of proteins increase with increasing salt concentration (salting in). As the salt concentration continues to increase, however, the solubility of the protein begins to decrease. At high ionic strength, the protein will precipitate out of the solution "salting out". When ammonium (NH4+) and sulfate (SO42−) ions are within the aqueous solution they are attracted to the opposite charges available on the protein that is being purified. Attraction of opposite charges prevents water molecules from interacting with the proteins This leads to precipitation (salting out)
Currently, many commercial (SUS) proteins start their purification using a process of ammonium sulfate flocculation. The salt ammonium sulfate is used to concentrate proteins Not destructive to the protein because aggregates of proteins form and individual protein structure is not disturbed.
Flocculation
Questions to consider Precipitation using strong acids over several hours A.
Preserve therapeutic protein structure and function
B. C.
Destroy therapeutic protein structure and function Have no effect on therapeutic protein structure and function
Questions to consider Weak cations such as PEI bind A.
Proteins
B. C.
NACL Both A & B
Questions to consider Salting out protocols can be used with _____________and prevent protein denaturation A.
Ammonium sulfate flocculation
B. C.
Alcohol precipitation Membrane precipitation
Questions to consider Flocculation is a common procedure used to A. B. C.
Precipitation soluble constituents in the cell harvest media Denature extraneous proteins Aggregate therapeutic proteins
Questions to consider _________ is the principal performance indicator used for the cell harvest precipitation step. A. B. C.
Packed cell volume pH Temperature
LAB
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Lab Module: The importance of acid for precipitating insoluble molecules from culture media; the importance of reversible flocculation of therapeutic proteins using ammonium sulfate •
Precipitation •
Citric acid precipitation of CHO cells in culture media prior to centrifugation and filtration • • •
•
1 M citric acid 4.0 60 minutes
Flocculation • •
•
Acid: pH: Time:
Ammonium sulfate flocculation of CHO cells directly from culture media prior to filtration Ammonium sulfate flocculation of media alone
Microscopic visualization
12:50pm-1:50pm
LUNCH – 60 MINUTES
75
1:50pm-3:20pm
CENTRIFUGATION
Courtesy of Amgen
Courtesy of Amgen 78
Centrifugation • •
Packed cell-serum separation Used in a number of bioprocess applications, including clarification of solutions from hormone, vaccine, and enzyme production; bacterial culture harvest, mammalian cell culture harvest and concentration and separation of blood fractions.
Centripetal Vs Centrifugal Forces: it’s your frame of reference Centripetal Force
Centrifugal Force
"the force that is necessary to keep an object moving in a curved path and that is directed inward toward the center of rotation,"
"the apparent force that is felt by an object moving in a curved path that acts outwardly away from the center of rotation" (Merriam Webster)
(Merriam Webster)
F-Force m-Mass v-velocity r-radius
Centripetal Vs Centrifugal Forces: it’s your frame of reference
Theory supporting centrifugation methods Particles are separated from the fluid stream by their sizes, shapes and densities The densities of the particles which may or may not be related to their size are separated by artificial (g) centrifugal forces Heavier particles/solids will sediment at the bottom of the system Basic terms: Pellet Supernatant Relative centrifugal force Critical speed
Centrifugation basics Velocity-rate and direction of an objects movement (m/s) Rate of sedimentation- s -(Svedberg) The rate of sedimentation depends on the density, size and shape of the molecule A 23S rRNA will sediment at a velocity of 23×10-12 m/s under normal gravity. In an ultracentrifuge producing an acceleration of one million g the velocity will proportionally scale to 23×10-6 m/s or about 1 mm/min.
What is density? Density - ratio of water (aqueous) content and dry mass (m/v) Water has a density = 1 and other molecules are relative to this Charged molecule are affected by surrounding shells of water and their densities are affected thus becoming more buoyant (buoyant densities) Effective densities Table 1: Densities of biological objects relative to water. This is almost equivalent to giving them in units of g/ml or 1000 kg/m3. Values are sorted in descending order. Unless otherwise stated, values were measured in sucrose or ficoll solution. (Harvard.edu)
Density gradients
Types of centrifuges that are used in biomanufacturing and general laboratory settings
Disk stack centrifuge
Swinging bucket: tabletop
Microcentrifuge
Key parameters for disc stack centrifugation in biomanufacturing Disc stack centrifugation / Separation G-force (gravitational) Discharge frequency of impurities Residence time under artificial g-forces Cell density Cell viability
GEA WESTFALIA (WWW.GEA.COM)
Disc stack centrifuge for bioprocessing Disc-stack centrifuges are fed continuously but discharge solids either periodically or continuously. A feed stream is fed into the centrifuge bowl while it rotates at a high g-force. Differences in density force cells to the periphery of the bowl, where they accumulate in the solids space, while cell-free centrate flows up the stacked discs through a centripetal pump and is collected for further processing. Centrate backpressure is applied to ensure hydrohermetic operation and that the centrifuge bowl remains full.
A more detailed view of disc stack centrifugation 1. 1. Product Feed 2. 2. Disc Stack 3. 3. Separating Discs 4. 4. Concentrate chamber
5. 5. Nozzles 6. 6. Centripetal pump for light phase 7. 7. Discharge of light phase 8. 8. Centripetal pump for medium phase 9. 9. Discharge for medium phase 10. 10. Feed for wash water or concentrate recycle
Disc-stack centrifuge for liquid/liquid/solid separation. Courtesy GEA Westfalia.
BioNetwork
Periodic Vs Continuous discharge centrifugation
Figure 1: GEA disc-stack centrifuge for periodic solids discharge BioProcess International by Ardarion Richardson and Joshua Walker Thursday, April 19, 2018
Figure 2: GEA disc-stack centrifuge for continuous solids discharge
Process Parameters
Courtesy of Amgen
Centrifugation calculation
93
Questions to consider What parameter affects centripetal force? A. Length B. Height C. Speed
Questions to consider When centrifuging cells and media during the cell harvest process, insoluble cellular constituents A. Always settled to the bottom of the centrifuge vessel B. May settle along with cells depending upon the centrifugal force C. Never settle to the bottom of the centrifuge vessel
Questions to consider Which type of centrifuge is most often used for cell separation processes in biomanufacturing? A. Disk stack centrifuge B. Swinging bucket centrifuge C. Microcentrifuge
Questions to consider During the centrifugation step, _____________ is pumped through a pump and further processed downstream. A. Isolated biological product B. Cells and centrate C. Cell-free centrate
LAB
2
Lab Module: The importance of density for centrifugal separation •
Centrifugation •
Percoll Density Gradient • • •
•
Media alone CHO cell suspension Acid precipitated CHO cell suspension
Microscopic visualization
3:20pm-3:40pm
BREAK – 20 MINUTES
2
3:40pm-5:00pm
FILTRATION
Courtesy of Amgen
Courtesy of Amgen 103
Filtration in biopharmaceutical manufacturing
Journal of Membrane Science Volume 620, 15 February 2021, 118804
Theory supporting filtration methods •
Separation of different size particles by passing through a membrane having a selective pore size.
Purpose of filtration in biopharmaceuticals •
Separation of soluble molecules from a solute by passing through a selectively permeable (semipermeable) membrane with a nominal molecular weight cut off
•
Clarification of the solute stream for preparation of downstream purification
Types of filtration Depth filtration Tangential flow filtration Alternating tangential flow filtration Precipitation and filtration Virus filtration Ultrafiltration/diafiltration Sterile filtration
Key points to consider during filtration separation Separator process (dead end, depth, cross flow) Type of filter membranes Filter membrane combinations Particle size distribution Turbidity profile Filter capacity
Structures of membrane materials
(A)
(B)
(C)
( D)
FIG. 14.5 Pictures on different material of construction and properties: (A) Nodular structure, (B) Open cell foam, (C) Macro-void structure, (D) Macro-void free structure.
Filtration Principles Jakob Liderfelt, Jonathan Royce GE Healthcare Life Sciences, Uppsala, Sweden
Types of membrane materials Sintered metal
Natural Cellulose
Polymeric Polyvinylfluorodyne (PVDF) Polyethersulfone (PES) Polytetrafluoroethylene (PTFE) Nylon
Open question: Which is best for an aqueous system?
Open question: How to make a hydrophobic membrane more hydrophilic?
Dead end filtration • • • •
Membrane with a defined pore size Batch process Cake layer formation Open question: Drawback?
https://assets.fishersci.com
Depth filtration
•
•
Filters a broad range of particles through pore size gradients Brittany Nixon, 3M Purification Nominal molecular weight cut off
What is depth filtration? Porous filtration that consists of decreasing pore sizes along the filter’s thickness. Retain particles that are smaller than its pores through a pore-size gradient that separates a broad range of particle sizes Particles can become trapped in the torturous path of the membrane PALL.com
Depth filtration system
https://www.pendotech.com/
Crossflow filtration/tangential filtration
What is micro-filtration (Tangential flow filtration)? Suspension flows at known rate of speed tangential (parallel and at an angle to) the filtration membrane As flow continues, solutes with molecular weights smaller than the membrane’s nominal pore size will traverse the membrane skin to the other side. Larger moieties will be inhibited from traveling through the pores. Barrier or fouling layer builds up along the length of the membrane eventually inhibiting filtration
What is micro-filtration (Tangential flow filtration)?
Academic Library
Molecular weight cut off
https://synderfiltration.com/
The importance of flow, pressure and resistance
A = V/J x T A = Membrane area (m 2 ) V = Volume of filtrate generated (liters) J = Filtrate flux rate (liters/ m 2/hour) T = Process time (hours)
Open question: Blood flow Vs Suspension flow: What affects pressure and resistance?
Amgen: Filtration Parameters
Courtesy of Amgen
Amgen: Filtration Parameters
Harvest filtration calculations
122
Harvest filtrate neutralization parameters
Courtesy of Amgen
Harvest filtrate neutralization parameters
Courtesy of Amgen
Harvest filtrate neutralization performance indicators
Courtesy of Amgen
Harvest filtrate neutralization performance indicators
Courtesy of Amgen
Neutralization calculations
Courtesy of Amgen
Raw material specifications materials for harvest
Courtesy of Amgen
LAB
129
Lab Module: The importance of filtration gradients (depth filtration) for separating waste from therapeutic antibody •
Graded filtration through 0.45µm and 0.22µm filters/depth filter • • •
Media alone CHO cell suspension Acid precipitated CHO cell suspension
TBD
5:00pm-5:15pm
WRAP UP
2
Take away………. •
•
•
You should understand the “whys” behind cell harvest processes including development and applications. You should have gained an understanding of the theories behind and methodologies of three basic cell harvesting techniques; precipitation/flocculation, centrifugation and filtration. You should understand concepts and applications for the three cell harvesting processes and thus have gained comprehensive training on current industry-based cell harvesting processes and standards including those specific to Amgen.
Thank you!