Introduction to Advanced Biopharmaceutical Purification Nov 22nd, 2021
Instructors
▪ Malcolm Pluskal, Ph.D., Landrau Scientific Innovations ▪ Sharon McGuire, MS, PDI, The University of Rhode Island
2
Advanced Chromatography Methods Development
3
Methods Development - Overview UPSTREAM Processing
Sample Matrix
DOWNSTREAM Processing
Define
Experiment
Evaluate
Implement
Optimize 4
Define Objective of Chromatography Process ▪ EXTRACT target molecule from sample matrix; ▪ ▪ ▪ ▪
Lyse cells and breakup cell walls Separate whole cells from sample Clarify by filtration (< 0.45 µm) of the sample Pre-sample preparation to remove contaminants
▪ CAPTURE a target molecule from a complex sample matrix; ▪ Increase target molecule concentration in the sample ▪ Remove bulk contaminants ▪ Stabilize target molecule from post-extraction degradation
▪ POLISH target molecule after capture to remove contaminants down to acceptable limits set by application; ▪ Limits set by FDA for therapeutic or diagnostic application ▪ Limits set by its use in an orthogonal process step
5
Molecular Characteristics Charge
- measure of the number of ionic charges on the surface of the molecule
Hydrophobicity
- measure of the hydrophobic character of the molecule
Affinity
- presence of sites on the molecule that can form high affinity interactions with other molecules
Solubility and stability
- ranges that a target molecule will stay in solution and maintain its biological activity
Molecular weight
- measure of size and shape of the target molecule
Understanding CHASM characteristics of a target molecule relative to other components in the sample matrix will aid in identifying properties that can be used to purify the target molecule away from other components. DEFINE a “purification handle” for capture and polishing of the target molecule 6
Types of Chromatography Type of Chromatography
Charge Exchange Hydrophobic Interaction
Affinity
MOA
Binding Conditions:
Net surface charge
Low ionic strength
High ionic strength; Increased (cation exchange) or decreased (anion exchange) pH
Hydrophobicity
High ionic strength
Low ionic strength
A bio-specific interaction leading to high affinity binding
NO competing ligand present
Competing ligand (specific); conditions that disrupt protein/protein interactions (non-specific)
Conditions that allow Low ionic strength at Solubility and Stability the target molecule to mid-point of pH stay in solution bioactivity
Molecular Weight
Elution Conditions:
Hydrodynamic radii (Stokes radius) and shape (asymmetry)
High concentration of ions and pH at which the molecule is denatured and can lead to aggregation
Separation based on Isocratic elution under optimal binding hydrodynamic size and conditions shape 7
How do you define molecular characteristics? ▪ Run an SDS-Page gel to display the molecular weight complexity of the sample matrix ▪ Run an Isoelectric gel to display the charge distribution of the sample matrix ▪ Combine in a 2D gel system if available ▪ Develop an assay to locate the target molecule of interest; ▪ Raise an antibody to the target and use a Western blot ▪ Carry out a screening assay in a multiwell plate format and measure bioactivity ▪ Use a rapid HPLC assay using an affinity ligand to titer the target molecule during the purification process ▪ Locate key information from web sites on the internet!
8
Human Serum 2D Gel pattern Vitamin D Binding Protein [ 53.8K, pI 5.2 ]
Alpha 2 Glycoprotein [ 50.3K, pI 4.5 ]
Haptoglobulin [ 47.1K, pI 4.7 ]
Apo A1 [ 23.1K, pI 5.2 ]
Transthryetin [ 13.8K, pI 5.5 ] MGP personal data
Alpha 1B Glycoprotein [ 73.2K, pI 5.0 ]
Complement Factor B [100 K, pI 6.2 ] Sero-Transferrin [ 75.6K, pI 6.4 ] Albumin [ 66.5K, pI 5.7 ]
Ig HG [ 52.3K, pI 8.3 ]
Ig LC [ 24.2K, pI 7.4 ] Haptoglobulin Alpha 2 [ 16.9K, pI 6.1 ]
9
Know your protein!
Human Serum Albumin
Primary Amino Acid Sequence MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPFDEHVKLVNEL TEFAKTCVAD ESHAGCEKSL HTLFGDELCK VASLRETYGMADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEFKADEKKFWGK YLYEIARRHP YFYAPELLYYANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLTSSARQR LRCASIQKFG ERALKAWSVA RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKECCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEKLGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLPDTEKQIKKQT ALVELLKHKP KATEEQLKTV MENFVAFVDK CCAADDKEACFAVEGPKLV WSTQTALA FEBS Letters 58, 134-137 (1975)
MWt. (molecular weight) = 69,000 Daltons (69 kD) pI (isoelectric point) = 5.82 Hydrophobicity index = - 0.395 https://en.wikipedia.org/wiki/Bovine_serum_albumin
10
Protein Chemistry – Charge distribution
http://www.mad-cow.org/00/annotation_frames/tools/genbrow/hgwdev.html
11
Protein Chemistry – Hydrophobicity
https://www.allometric.com/tom/courses/bil255/bil255goods/03_proteins.html
J. Mol. Biol. 157:105-132(1982)
http://www.mad-cow.org/00/annotation_frames/tools/genbrow/hgwdev.html
12
Protein chemistry - MWt. distribution
http://www.mad-cow.org/00/annotation_frames/tools/genbrow/hgwdev.html
13
Ion Exchange (IEX) Chromatography
https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/0471142727.mb1010s44
14
Direct Capture by Cation IEX
http://www.mad-cow.org/00/annotation_frames/tools/genbrow/hgwdev.html
https://www.sartorius.com/download/483882/ceramic-hyperd-datasheet-en-b-2579910-sartorius-data.pdf
15
Hydrophobic interaction (HIC) HIC ligands
https://www.researchgate.net/figure/The-hydrophobic-ligand-protein-interaction fig1_304237360
Hoffmeister Series of Counter ions
https://www.bio-rad.com/en-us/applications-technologies/ introduction-hydrophobic-interaction-chromatography-hic?ID=MWHB53MNI
16
Mixed Mode Chromatography
https://www.cytivalifesciences.com/en/de/solutions/protein-research/knowledge-center/protein-purification-methods/Multimodal-chromatography
https://www.jnc-corp.co.jp/fine/en/cellufine/grade/grade-8/
17
Affinity Chromatography
18
Size Exclusion Chromatography (SEC)
https://www.creative-proteomics.com/pronalyse/size-exclusion-chromatography-sec-service.html
19
Break
20
Development of a Purification Workflow Computer modelling development of a multi-step purification workflow ▪ Two step purification of a basic protein by anion exchange capture followed by size exclusion chromatography polishing ▪ Three step purification of an acidic protein by cation exchange capture followed by hydrophobic interaction chromatography
21
Purification of a Protein from a Complex Mixture by Anion Exchange Chromatography
Video 1
22
Protein Purification Optimization of a Cation Exchange Resin
Video 2
23
Trends in Bioprocess
24
Bead types improvements in mass transport
25
Fibrous Membrane Absorbers improvements in mass transport, flow rate but still lower capacity?
https://www.lobov.com.ar/downloads/Instructions-HiTrap-HiScreen%20Fibro%20PrismA.pdf
26
Continuous Bioprocessing
https://iscmp2014.mit.edu/sites/default/files/documents/ISCMP%202014%20White%20Paper%204%20-%20Continuous%20Bioprocessing.pdf
27
Multicolumn Bioprocessing
28
Lab 1 Purification of Green Fluorescent Protein (GFP)
29
What is Green Fluorescent Protein? Green fluorescent protein (GFP) is a protein in the jellyfish Aequorea Victoria that exhibits green fluorescence when exposed to light. The protein has 238 amino acids, three of them (Numbers 65 to 67) form a structure that emits visible green fluorescent light. In the jellyfish, GFP interacts with another protein, called aequorin, which emits blue light when added with calcium. 3D Structure GFP Excitation-Emission Spectra Size – 28 kDa pI – 5.58 Hydrophobicity - High
30
Objective of Lab 1 Recover GFP from a mixture; ▪ Lysozyme (pI 11.35) and Oligo dT 40 bp (pI < 2.0)
▪ Polyclonal antibody (pI 7.4 – 9.2) and Oligo 40 bp (pI < 2.0) ▪ Using an Anion exchange cartridge on an AKTA Purifier + fraction collector ▪ Loading buffer 50 mM Tris HCl pH 8.5 ▪ Elution with a gradient up to 0.5M ▪ Collect 0.5 mL fractions https://remotedesktop.google.com/?lfhs=2
▪ Discussion of result? ▪ How can we improve the above separation?
31
Summary of GFP Methods Development Mini Column 6 7mmxDx3cmL001:10_UV1_280nm Mini Column 6 7mmxDx3cmL001:10_Cond
Mini Column 6 7mmxDx3cmL001:10_UV2_395nm Mini Column 6 7mmxDx3cmL001:10_Fractions
Mini Column 6 7mmxDx3cmL001:10_UV3_260nm Mini Column 6 7mmxDx3cmL001:10_Conc@06,SHFT3
mAU
1400
1200
Lysozyme
GFP
1000
800
Oligo dT 600
400
200
1
0 0.0
2 2.0
3
2
1 4.0
4
3 6.0
6
5 8.0
7
Waste
8 10.0
12.0
14.0
16.0
ml
32
GFP Purification from a Polyclonal Ab and an Oligonucleotide
Lab 1 GFP Purification
33
Summary of GFP + Antibody Methods Development Mini Column 6 7mmxDx3cmL001:10_UV1_280nm Mini Column 6 7mmxDx3cmL001:10_Cond@04,SHFT
Mini Column 6 7mmxDx3cmL001:10_UV2_395nm Mini Column 6 7mmxDx3cmL001:10_Inject
Mini Column 6 7mmxDx3cmL001:10_UV3_260nm Mini Column 6 7mmxDx3cmL001:10_Logbook
mS/cm
80.0
GFP
70.0
Oligo dT
60.0
50.0
40.0
30.0
Polyclonal Ab Re_Equilibrate
20.0
Elution
Wash
10.0
0.0 0.0
5.0
10.0
15.0
20.0
25.0
min
34
Observations – What have we learned? ▪ Nucleic acids are strongly retained on an AEX resin ▪ GFP can be monitored at its extinction wavelength of 395 nm ▪ Highly basic proteins (with a high pI) such as lysozyme are not retained by an AEX resin at pH 8.5 ▪ Polyclonal antibodies are weakly retained by the AEX resin at pH 8.5 due to their basic pI range
35
Assessment 1
36
Lunch
37
Protein A Affinity Methods Development
38
Historical Overview – Biological Role As a pathogen, Staphylococcus aureus (SA) utilizes Protein A, along with a host of other proteins and surface factors, to aid its survival and virulence. To this end, Protein A plays a multifaceted role: ▪ By binding the Fc portion of antibodies, Protein A renders them inaccessible to the cells response mechanism to a foreign cell, thus impairing phagocytosis of the bacteria via immune cell attack ▪ Protein A has been shown to cripple humoral (antibody-mediated) immunity which in turn means that individuals can be repeatedly infected with S. aureus since they cannot mount a strong antibody response ▪ Protein A has been shown to promote the formation of biofilms both when the protein is covalently linked to the bacterial cell wall as well as in solution ▪ Mutants of SA lacking Protein A are more efficiently phagocytosed in vitro, and mutants in infection models have diminished virulence ▪ Free Protein A has been reported to act a pyrogen in very low levels 11/17/2021
Proprietary to FloDesign Sonics
39
39
Protein A Structural Biology Protein A is a 42 kDa surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. It is encoded by the spa gene. spa gene organization N
C Signal peptid e
Trans-membrane insertion sequence
FC binding domains for antibodies
Are all the binding domains equivalent? YES/NO ▪ B domain shows the least substitutions – consensus sequence ▪ Overall Kd for FC binding 5 nM (10-9) ▪ Individual domains in the range 10-100 nM – cooperative binding kinetics ▪ Domain E shows reduced binding ▪ Highest molar stoichiometry (IgG/Protein A) seen is 2.5 - steric hindrance? 40
40
Protein A Structure Staphylococcal Protein A (SPA), Z Domain Z domain of SpA is an engineered analogue of the IgGbinding domain B. The binding domain contains three alpha helices which are arranged in an antiparallel three-helix bundle. Four types of side chains are visible however: lysine (K)=blue, phenylalanine (F)=aqua, leucine (L)=green, and aspartic acid (D)=red. This domain of the protein contains no disulfide bonds and is 72 amino acid residues long.
41
41
Protein A Interaction with Immunoglobulin G
Fab
FC Protein A Binding Site - 1
Protein A Binding Site - 2 42
42
Bioprocess Purification with Protein A Sources of Native Protein A; ▪ From the original Staphylococcus aureus by extraction from the cell wall ▪ From a mutant that secreted the SPA into the cell culture (Fermentech) ▪ Recombinant gene expressed in Escherichia coli by Repligen; ▪ Original IP covering expression of whole SPA gene in E coli ▪ Purified on an IgG affinity resin ▪ Expired in 2012 ▪ Granted IP covering expression of truncated form of SPA gene in E coli ▪ More stable expression ▪ Purification on a wide range of expression based affinity ligands including IgG ▪ Expires in 2030 43
43
Bioprocess Purification with Protein A Next generation rProtein A molecules for Bioprocess Applications ▪ Improved base stability – remove base labile amino acids, such as Asparagine ▪ from the sequence to inhibit fragmentation of the polypeptide ▪ Improve the immobilization efficiency – use site directed immobilization strategies (via cysteine or lysine amino acids) to orientate the ligand on the affinity surface
▪ Reduce ligand leaching – more stable linkage to affinity support ▪ Increase ligand utilization efficiency for higher capacity – minimize steric hindrance on binding and increase the number of binding domains ▪ Improve elution of IgG – use higher pH milder conditions ▪ Increase protease resistance – removal of obvious protease cleavage sites 44
44
Bioprocess Purification with Protein A Sources of “next generation” engineered rProtein A; ▪ GE Healthcare – Mab Select SuRE ligand family ▪ Engineered B/Z domain with Asparagine's removed to improve base stability ▪ Tetramer construct 4 binding domains ▪ Orientated immobilization via a single engineered cysteine residue ▪ NHS chemistry for immobilization
45
45
Bioprocess Purification with Protein A Sources of “next generation” engineered rProtein A; ▪ Proteonova, Japan – R25 and R40 ligands (TOSOH)
▪ Engineered C domain to improve base stability ▪ Hexamer construct 6 binding domains ▪ Orientated immobilization via lysines engineered in-between binding domains ▪ Reductive amination chemistry for immobilization ▪ Improved ligand utilization
46
46
Protein A Workflows for IgG Purification Conventional batch column processing; ▪ ▪ ▪ ▪ ▪ ▪
Resin packed in a column Pre-packed columns are available Hydrogel membrane in a cartridge (Sartorius, Natrix) Batch process loading to 80% of dynamic binding capacity Discontinuous process Uses available hardware
Alternative workflows; ▪ ▪ ▪ ▪
Use multiple columns Amenable to pre-packed column use Load to full resin capacity Continuous output of product linked to a perfusion bioreactor ▪ Simulated moving bed (SMB – Tarpon/Pall) ▪ Periodic Counter Current (PCC – GE) ▪ Requires complex hardware and in-process analytical testing for control feedback 47
47
Bioprocess Purification with Protein A New Bioprocess Workflows with rProtein A molecules - Continuous downstream purification • • • •
Multiple smaller columns, Complex hardware, Resin compatible with high flow processing, Full utilization of the resin binding capacity Requires rapid on-line process monitoring, • Continuous product output.
11/17/2021
Proprietary to FloDesign Sonics
48
rProtein A Affinity Capture
49
Capture-polishing process integration - Protein A capture followed by IEX and mixed mode polishing
50
Overview of AMGEN multistep purification process - Order of the chromatographic processes may change depending On protein being purified. Typically, purification begins with Protein A chromatography
Courtesy of Amgen 51
Overview of AMGEN multistep purification process for a mAb – Capture from cell culture
52
Overview of AMGEN multistep purification process for a mAb - Polishing ▪ Polishing Step 1 – low pH viral inactivation ▪ Polishing Step 2 – Mixed mode AEX (Capto Adhere MMC); ▪ ▪ ▪ ▪ ▪
157 L column volume mAb Flow Through mode High mAb recovery (> 80%) Slight increase in volume Removes CHO-HCP, dsDNA, leached Protein A, mAb aggregates, viruses and endotoxin
▪ Polishing Step 3 – Cation Exchange (Fractogel-S CEX) chromatography; ▪ 308L column volume ▪ Bind and elute mode ▪ Removes mAb dimers, aggregates and CHO-HCP
▪ Polishing Step 4 – Viral filtration ▪ UF/DF to condition the final mAb product for formulation 53
Lab 2 Purification of a mAb by Affinity Chromatography
54
Objective of Lab 2 Capture a mAb from a CHO cell culture supernatant; ▪ mAb titer 0.36 mg/mL clarified CHO cell supernatant, ▪ Using 1 mL rProtein A affinity cartridge on an AKTA Purifier + fraction collector, ▪ Loading buffer 25 mM Tris HCl and 150 mM NaCl pH 7.5, ▪ Elution with a step gradient with 60 mM Na Acetate pH 3.5, ▪ Collect 10 mL fractions of the flow through,
▪ Pool elution fraction and neutralize with 1 M Tris base.
https://remotedesktop.google.com/?lfhs=2
55
Process Analytical Monitoring Rapid measurement of mAb titer in purification fractions; ▪ Use a POROS ProA analytical affinity column (Thermo-Fisher), ▪ Loading buffer 25 mM Tris HCl and 150 mM NaCl pH 7.5, ▪ Elution with a step gradient to 12 mM HCl, ▪ 2 mL/min flow rate, ▪ Measure elution peak area at 1.25 min ▪ 3 min total analysis time, ▪ Use a mAb standard curve to estimate the titer.
https://remotedesktop.google.com/?lfhs=2
56
mAb capture by ProA from CHO cell supernatant
57
Process Monitoring of mAb during Capture Step CHO mAb capture R1 10192021001:10_UV1_280nm CHO mAb capture R1 10192021001:10_Logbook
CHO mAb capture R1 10192021001:10_pH
CHO mAb capture R1 10192021001:10_Fractions
mAb
mAU
4000
160.0 140.0 3000
120.0
Peak Area
100.0 2000
10% DBC Batch load
80.0 60.0
0.0
F2
0
0
1
2
3
5
4
5 50
6
7
8
10
9
10
11
12
100
13
15
14
15 150
16
17
18
20
19
20 200
21
22
23
25
Elution
Wash Down after Load
0
Equilibration Sample Load
20.0
F3
Re Equilibrate
1000
40.0
Waste ml
Fraction Number
58
Observations – What have we learned? ▪ rProtein A can capture mAb directly from cell culture clarified supernatant, ▪ Rapid in-process mAb titer monitoring can be used to determine process load parameters, ▪ Retained mAb can be rapidly eluted in 2-3 column volumes by lowering the pH to 3.5.
59
Assessment 2
60
Break
61
Formation and Removal of mAb Aggregates
62
Protein Aggregation ▪ Clinical in vivo aggregation ▪ Alzheimer's – amyloid fibrils ▪ Parkinson’s disease ▪ Up to 20 diseases have been reported to be due to protein aggregation
▪ Aggregation during bioprocessing ▪ Cell culture ▪ Protein purification ▪ Biologics formulation and storage
63
What is an aggregate? Soluble/insoluble ▪ Soluble – not visible under microscope and pass-through a 0.2 µM microporous filter ▪ Insoluble – retained by a filter and in the size range 10-25 µM in size
Covalent/non-covalent ▪ Formation of a chemical bond between 2 or more molecules ▪ Disulphide bonds ▪ Oxidation of Tyrosine's
Reversible/non-reversible ▪ Non-covalent interactions can be reversible
64
How do aggregates form? Thermal induced process
Freeze and Thaw induced changes
Mechanical induced changes
Nucleation induced rapid generation of aggregates – exponential kinetics Nucleation followed by monomer elongation of aggregates
Linear increase with time
65
Aggregation in cell culture ▪ Over-expression of proteins can lead to formation of insoluble inclusion bodies – protein crystalline aggregates, ▪ mAb’s in CHO cell culture can have unpaired –SH groups which can lead to covalent bond formation, ▪ Increase in protein product concentration during culture can lead to aggregate formation.
66
Aggregation during purification ▪ To achieve the high level of purity required, multiple orthogonal purification techniques are employed – IEX, Protein A, HIC etc., ▪ Process exploits differences in biospecific affinity, charge, size, or other properties of the desired protein from the impurities to enhance purification, ▪ Protein experiences a wide range of pH, ionic strength, oxidation/reduction and protein concentrations during the purification.
Each condition experienced by the protein may affect the degree of aggregation observed 67
Aggregation during UF/DF processing ▪ Ultrafiltration/diafiltration (UF/DF) is typically performed to exchange the buffer and to increase the protein concentration in solution – later stage formulation ▪ UF membrane fouling due to the formation of aggregates in the region of locally high concentration – gel layer “membrane” ▪ During the UF/DF process the protein is being continually pumped, with a typical process requiring at least 50 passes through the pump – mechanical shear stress must be controlled
68
Aggregation during filling Rolling diaphragm pump vs. a radial piston pump? In the piston pump the protein solution is used to lubricate the piston during the operation – source of shear stress
69
Removal of Particulates/Aggregates ▪
Depth filtration with MF membranes; ▪ ▪ ▪ ▪
▪ ▪ ▪
Whole cells Cell debris Heterochromatin (DNA + protein) Final pass through at 0.22 µM sterilizing filter
Ultrafiltration membranes SEC chromatography Polishing chromatography: ▪ ▪ ▪ ▪ ▪
Bind and elute on high performance IEX Flow through on a mixed mode media Hydroxyapatite mixed mode Affinity chromatography HIC 70
Measurement of Aggregates ▪ ▪ ▪
SEC chromatography – size-based separation Dynamic light scattering – particle size distribution Analytical Ultracentrifugation – sedimentation analysis
▪
Aggregate specific fluorescent dye binding ▪ ▪ ▪ ▪
High throughput Multiwell plate based High sensitivity Comes with aggregation standards
71
PROTEOSTAT® Protein aggregation assay Dye is immobilized when bound to the aggregate and begins to fluoresce.
Excitation
Emission
Effective linear dynamic range for antibody aggregate detection using PROTEOSTAT® Detection Reagent compared with Thioflavin T. Relative fluorescence unit values (RFUs) may differ depending upon the microplate reader employed for the analysis.
72
Lab 3: Analysis of mAb Aggregates by Size Exclusion Chromatography (SEC) 73
Lab 3 Analysis of mAb Aggregates by Size Exclusion Chromatography (SEC)
74
SEC Analysis of mAb Aggregates ▪ TSKgel Super SW mAb HR column, 4 μm, 7.8 mm ID × 30 cm ▪ Mobile phase 0.2 M Na Phosphate, 0.1M Na Sulfate pH 6.7 ▪ Flow rate 0.8 mL/min ▪ Run time 20 min ▪ Detection at 280 nm ▪ Injection volume 20-50 µL
75
SEC Calibration Curve ▪ Calibration Curve 7.000
Log10 Molecular Weight
▪ Gel Filtration Standard (P/N 1511901, Bio-Rad)
6.000 5.000
4.000 3.000 2.000 1.000
0.000 4.00
6.00
8.00 10.00 12.00 Retention Time (min)
14.00
16.00
76
Low pH/Heat Induced mAb Aggregate formation ▪ mAb elution fraction from rProtein A capture ▪ Adjust concentration to 2-3 mg/mL by dilution in a pH 5.0 Acetate buffer ▪ Heat at 60oC for 2 to 20 min ▪ Cool to room temp ▪ Store samples at 2-8oC in HPLC auto-sampler ▪ Analyze by SEC
77
Time Course of Heat Denaturation Control mAb 2 min at 60oC 8 min at 60oC 6000 670 kDa kDa
mAb DIMER 310 kDa
mAb MONOMER 155 kDa
12 min at 60oC 20 min at 60oC 6000 670 kDa kDa
310 kDa
155 kDa
78
Observations ▪ Initial rProtein A captured mAb did show some aggregated material ▪ Heating led to loss of the monomer peak ▪ Large increase in high molecular weight fraction ▪ Aggregate peak slightly > in size than the Blue Dextran high molecular weight void volume (Vo) standard! ▪ Prolonged heating leads to the formation of visible aggregates and the protein suspension turns turbid
79
Assessment 3
80
Thank You!
Contact Information: Malcolm Pluskal, Ph.D. (email: m.pluskal@verizon.net) Sharon McGuire, MS, (email: smcguire@uri.edu)
81
Wrap Up and Evaluations
82