Purification of a Monoclonal Antibody Using the
Ionela Iliescu1, Robert S. Gronke1,
1
Purification Development, Biogen Idec Inc, 14 Cambridge Center, Cambridge, MA 02142, USA
Abstract MAbsorbent Ligands Shorter Spacer Arms Improved IgG
Binding Capacity of A2P
The affinity ligand Protein A is a powerful tool to purify monoclonal antibodies. Though
Protein A Binding Site
widely used by the Biotech industry, Protein A has several shortcomings including its high
cost ($9,000 鈥? $12,000/Liter), instability to strong base (i.e.1 N NaOH), leaching (ligand is
Resin))
toxic) and for some monoclonal antibodies, elution at low pH (< 3.5). ProMetic BioSciences,
Binding Capacity (mg IgG/mL Resin)
25
in collaboration with Biogen Idec Product Development have developed a small molecule
A1P
affinity ligand called A2P that has been shown to be competitive to protein A. The affinity
ligand was designed using combinatorial synthesis, involvig controlled substitution to a 20
triazine ring. Immunoglobulin binds to A2P in low salt through a hydrophobic interaction
mechanism. The antibody binding capacity was found to be a function of ligand density and
Phe Tyr
length of the spacer arm used. Pluronic F-68, a frequently used mammalian cell culture 15
shear protectant, was found to interfere with antibody binding to the synthetic affinity ligands.
Thus, the cation exchanger Fractogel SO3- (EMD) was inserted as a first purification step to
remove the Pluronic F-68. 10
MAbsorbent庐 A2P resin and a related synthetic affinity resin, MAbsorbent庐 A1P, also A2P
5
developed by ProMetic BioSciences were evaluated using a mammalian cell culture
feedstream containing a monoclonal antibody (mAb1) produced at Biogen Idec. After
capture on Fractogel SO3- the product was directly loaded onto MAbsorbent庐 A1P or A2P
0
adsorbents. The product was eluted off the column using low pH. Both synthetic affinity
adsorbents performed similarly, resulting in good yields and >95% purity based on reduced 0 2 3 4 6 8 12
gel chip analysis. An alternative elution condition using 60% ethylene glycol (EG) at neutral
Mimic Spacer Arm Length (# Carbons)
pH was also used for comparison. The majority of mAb1 was eluted off A1P and A2P in the
60% EG fraction. The preliminary results suggest that the elution of the A2P resin with 60%
EG may provide product with superior yield and purity.
Objectives In General, Increasing Ligand Density
Combinatorial Synthesis Involves
Improved Binding Capacity of A2P
Controlled Substitution to a Triazine
Binding Capacity (mg IgG/mL Resin)
Develop an affinity column specific for humanized IgG expressed in mammalian cells
as an alternative to protein A Triazine structure is first coupled
Reduce process purification cost 2 Carbon 3 Carbon 6 Carbon
40
to resin
Beta test the monoclonal antibody kit provided by ProMetic BioSciences with an
35
industrial feedstream 1st functional group is attached
Optimize kit procedure, if necessary, to improve purification results
under mild conditions 30
25
2nd functional group is attached
Cl
under more stringent conditions 20
Methods
N 15
10
Reduced and non-reduced SDS-PAGE
Agarose N
X
Reduced and non-reduced gel chip 5
Size exclusion chromatography
0
Protein A HPLC assay for titer determination
0 5 10 15 20 25 30 35 40
N
TCID50 assay for Mice minute virus (MMV) and Xenotropic Murine leukemia virus
Cl Ligand Density (碌moles epoxide/g)
(X-MLV)
Triazine Structure
F-68 (cell culture media component)
Varied Affinity Column Components
Evaluation of Antibody Capture Step
Reduced the Binding Capacity of IgG to
the A2P Resin
Variable length # / bead
Adsorbent Type Advantages Disadvantages
Protein A Very good yield and Biological product (protein ligand)
35
high purity Difficult to clean/sanitize
IgG Binding Capacity (mg/mL)
Pure IgG
Good capacity Prone to degradation (leakage)
30
Reasonable lifetime May harbor endotoxin
Works for many mAb鈥檚 Expensive (>$9000/Liter) 25
Clarified Media
20
Synthetic Affinity Easy to clean/sanitize Lack of commercially available choices Pluronic F-68 (n = 140)
15
Ligands Low cost ($1000-3000/Liter) Little regulatory experience
High capacity (up to 50 mg/ml)
10
Long lifetime
Good purity 5
Agarose Spacer Arm Ligand
Works for many mAb鈥檚
0
0 0.5 1 1.5 2 2.5
Concentration of F-68 (g/L)
ProMetic BioSciences Monoclonal Antibody Kit
Jim Pearson2, and Keith Watson2
2
ProMetic BioSciences Ltd., 211 Cambridge Science Park, Milton Road, Cambridge CB4 0ZA, UK
Elution of A1P and A2P with Low pH
Replacement of Protein A with Mabsorbent庐 Virus Clearance on A2P Using 60%
鈥1P and A2P Eluates have similar purity with low pH elution
Cell Culture Suspension
Ethylene Glycol Elution (pH 7.8)
鈥owever, the purity can be further improved
Harvest Columns were eluted with pH 3.5 (elution 1), 3.0 (elution 2) and 2.0 (strip).
Eluates analyzed by reduced SDS-PAGE.
A1P A2P
Clarified Supernatant
Virus Removal Inactivation Total
Clearance
(log10) (log10)
(log10)
Cation Exchange Chromatography
Protein A Chromatography
MMV 4.9 <1 4.9
MAbsorbent Chromatography
X-MLV > 3.7 2.0 > 5.7
Further purification/Formulation
pH 3.5 pH 3.5
Elution Elution
Kit Strategy for Purification of Elution of A1P, A2P and rProtein A at pH 3.5
Conclusions
Monoclonal Antibodies
% Total Half Antibody by NR Gel Chip
Development of synthetic affinity resin
20 17.5
A 鈥渉alf antibody鈥?
Clarified Culture Supernatant
Optimized spacer arm length and ligand density increased the binding
variant was cleared
15
capacity of A2P with pure IgG
more efficiently by
10.7
Adjust to pH 6 9.8 Fractogel EMD SO3- used as the capture step if feedstream contains
9.5 the synthetic affinity
B
10
A pluronic F-68
ligands compared to
rProtein A resin. Evaluation of synthetic affinity resins (A1P and A2P) for purification
Capture with Fractogel EMD SO3- 5
of mAb1
(cation exchange)
0
Lower pH of clarified cell culture supernatant required for efficient
IEX A1P Eluate A2P Eluate rPA Eluate
capture of mAb1 on Fractogel EMD SO3-
Elute with 0.5 M NaCl
Evaluation of alternative elution conditions: low pH or 60% ethylene
% Aggregate by SEC
glycol at neutral pH
Purification with MAbsorbent庐 A1P/A2P 25
Preliminary results indicate that elution of A2P with 60% ethylene
20.4 Aggregate level with
glycol at neutral pH may provide superior yield and purity
C D
20
A2P or A1P was
Elute with low pH 60% EG elution step gave good clearance of MMV and X-MLV
much lower than that
15
of rProtein A eluate
9.4
10
Product/Analysis Cost analysis: two-step non-Protein A purification was
4.1 4.0
5
approximately 50% the cost of Protein A process
0
IEX A1P Eluate A2P Eluate rPA Eluate
Alternative Elution: 60% Ethylene Glycol
Results 鈥? Purification of mAb1
Elution, pH 7.8
鈥oor capture of mAb1 on Fractogel SO3- at pH 6.0
Acknowledgments
鈥apture of mAb1 improved at pH 4.7 Higher product yield and purity with A2P than with A1P
Fractogel 鈥? Load at pH 4.7 Columns were eluted with 60% EG (pH 7.8) followed by elution with pH 3.5 (elution 1),
Biogen Idec Inc. Prometic BioSciences Ltd.
3.0 (elution 2), and 2.0 (strip). Fractions analyzed by reduced SDS-PAGE.
Douglas Cecchini, Ph.D. Dev Baines, Ph.D.
A1P A2P
J枚rg Th枚mmes, Ph.D. Michael Duell
Anisa Vaidya Peter Bonnett
Christine Poliks Steve Burton
Removal of
Loss of impurities
Product
Primedica
Light Light
Chain Loss Chain Loss
Kate Bergmann
60% EG Eluate
60% EG Eluate
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