High throughput isolation of individual
glycoproteins (affinity SPE tools)
Jana Vidič
BIA Separations d.o.o
Split, 2nd of October 2013
WP2 – affinity SPE tools
Development of 96 well monolithic plates for
isolation of individual plasma proteins - with
immobilizied mAbs (delivery date 48 month) :
Transferrin, Fibrinogen, Haptoglobulin and Alpha
1-acid glycoprotein
BIASep tasks – WP2
o Activation of the monolithic support for further immobilization
o Development of the immobilization procedure and testing of
the imobilized antibody (a comercial monoclonal antibodies was
used)
o Production of the ordered anti-transferrin-mAb and antifibrinogen-mAB at the final stage (from MEDRI and Biotechnical
faculty Ljubljana)
o Immobilization of anti-transferrin and anti-fibrinogen on
monolithic columns
Activation of the matrix for further imobilization
- literature - beadso Affinity-purified antibodies conjugated to Hydrazide matrix
Affinity separation and enrichment methods in proteomic analysis. X. Fang, W. Zhang, J. Proteomics 71
(2008 ) 284 – 303
Immunoaffinity separation of plasma proteins by IgY microbeads: Meeting the needs of proteomic
sample preparation and analysis. Huang L. et al., Proteomics 2005, 5, 3314–3328
o Antibody immobilization using heterobifunctional crosslinkers
Affinity separation and enrichment methods in proteomic analysis. X. Fang, W. Zhang, J.
Proteomics 71 (2008 ) 284 – 303
o antibodies are attached to polymeric material through protein
A or protein G cross-linking
Multi-component immunoaffinity subtraction chromatography: An innovative step towards a
comprehensive survey of the human plasma proteome. Pieper R. et al., Proteomics 2003, 3, 422–432
Activation of the monoliths
Activation:
o CDI monoliths were used as received from production
o Aldehyde activation
o Glutaraldehyde (GA) immobilization to EDA and NH3 columns
o Hydrazide activation
o pA crosslinking
Monolithic materials:
o CIM epoxy columns
o CIM EDA columns
o CIM CDI columns
o CIM OH columns
o CIM NH3 columns
Hydrazide coupling
- Testing procedureo Hydrazide monoliths were prepared with different modification
solution from adipic dihydrazide; with and without hydrolysis
afterwards
o FTIR spectra, mass increase and elemental analyses were
followed in each step to determine the optimal modification
procedure.
o The BCA test was additionally used to prove the presence of
hydrazide groups on the monolith surface.
o hydrazide monoliths were tested for pA immobilization
Hydrazide coupling
- Results 1o IR spectra confirmed the remaining IR groups and an additional
broad band around 1600 cm-1, which belongs to hydrazide
carbonly groups.
epoxy
0,3
epoxy
hydrazide - before hydrolysis
hydrazide - after hydrolysis
Absorbance
0,2
hydroxy
0,1
carbonyl
3500
3000
2500
2000
1500
1000
-1
Wavenumber (cm )
Figure : FTIR spectra (recorded in ATR mode) of hydrazide monolith before and after the hydrolysis
o Max conversion of epoxy groups was around 10%.
Hydrazide coupling
- Results 2o BCA test of the final monoliths: the formation of coloured solution
with hydrazide monoliths proved the presence of hydrazide groups,
while blank tests (with NH3 monolith and epoxy monolith) did not
result in any colour formation
o testing for DBC for polyclonal IgG (Octagam) and a pronounced
capacity for the IgG was measured.
Table: IgG DBC values of hydrazide-GA-recombinant pA monoliths
Modification
Ionic capacity
IgG capacity (mg/ml of support)
procedure
Before/after hydrolysis
Before/after hydrolysis
acetate, pH 3.3
0 / 27
carbonate, pH 11
0 / 52
borate
0 / 41
7.8 / 6.6
7.76 / 3.4
7.78 / 7.0
As testing procedure for the activated
monoliths and determination of the
immobilization procedure a commercial
monoclonal antibodies was used.
The purpose was the immobilization of the monoclonal
antibody adalimumab on the monolithic support using
different immobilization procedures and testing the dynamic
binding capacity and selectivity of prepared monoliths towards
tumor necrosis factor alpha (TNF), which is an antigen of
adalimumab.
commercial mAb - CDI coupling
Table: Dynamic binding capacity of mAb-CDI affinity columns
Loading buffers
DBC value (mg antigen per
ml of chromatographic
support)
Elution area at 280
nm
Blank
0.05
6
mAb, MES buffer
0.05
22
mAb, phosphate buffer
0.05
5
As before, blank columns do not have any interaction with the columns, but even the
affinity columns have very limited affinity towards the antigen.
commercial mAb - EDA coupling and
Glutaraldehyde (GA) immobilization
Table: Dynamic binding capacity of mAb-GA-EDA affinity columns
Loading buffers
DBC value (mg antigen per
ml of chromatographic
support)
Elution area at 280
nm
Blank
0.05
5
mAb, MES buffer
0.05
8
mAb, phosphate buffer
0.19
12
As before, blank columns do not have any interaction with the columns. In case of
EDA-GA affinity columns, the affinity of the mAb towards antigen was almost
negligible. It seems that there is a small affinity of the column, prepared in phosphate
buffer.
commercial mAb - Aldehyde coupling
Table: Dynamic binding capacity of mAb-aldehyde affinity columns
Loading buffers
Blank
DBC value (mg antigen per
ml of chromatographic
support)
Elution area at 280
nm
0
5
mAb, MES buffer
0.31
51
mAb, phosphate buffer
0.29
37
Blank columns do not have any interaction with the columns, therefore it is
anticipated that theDBC value of a mAb columns results mainly from affinity
interaction between mAb and antigen. The affinity of the adalimumab columns
was proven, although the DBC values are not high (around 0.3 mg/ml). The
difference between two different immobilization buffers is not evident.
commercial mAb - NH3 coupling and
Glutaraldehyde (GA) immobilization
Table: Dynamic binding capacity of mAb-GA-NH3 affinity columns
Loading buffers
DBC value (mg antigen per
ml of chromatographic
support)
Elution area at 280
nm
Blank
0.05
8
mAb, MES buffer
0.29
52
mAb, phosphate buffer
0.33
64
Blank columns do not have any interaction with the columns, therefore it is
anticipated that theDBC value of mAb columns results mainly from affinity
interaction between mAb and antigen. The affinity of the adalimumab columns
was proven, although the DBC values are not high (around 0.3 mg/ml). The
difference between two different immobilization buffers is not evident.
commercial mAb – hydrazide activation
Table: Dynamic binding capacity of mAb-hydrozide affinity columns
Columns
Blank
Oxidized mAb, hydrazide
from acetate buffer
Oxidized mAb, hydrazide
from borate buffer
DBC value (mg antigen per
ml of chromatographic
support)
Elution area at 280
nm
0
4
0.74
80
0.65
70
Blank columns do not have any interaction with the columns, therefore it is
anticipated that theDBC value of adalimumab columns results mainly from
affinity interaction between mAb and antigen.
commercial mAb – pA crosslinking
Table: Dynamic binding capacity of mAb-pA affinity columns
Columns
DBC value (mg antigen per
ml of chromatographic
support)
Elution area at 280
nm
Blank
0.03
6.5
mAb, 1
0.81
135
mAb, 2
0.82
119
These results are the most promising of all tested mAb columns, because the DBC
value is the highest (around 0.8 mg/ml) and the breakthrough curve is steep (see
Figure). Consequently the elution of antigen from the column is more than twice
higher than from the columns with the capacity around 0.3 mg/ml, confirming a
reversible binding. As before, blank columns do not have any interaction with the
columns.
commercial mAb – pA crosslinking
blank
adalimumab column 4
Figure: Dynamic binding capacity measurements
of antigen over pA-mAb crosslinked monoliths
400
A at 280 nm
loading
elution
200
0
0
2
4
t (min)
From the ratio between the immobilized amount of mAb and the dynamic binding
capacity for antigen the number of active mAb molecules was calculated. The molar
weight of a mAb is app. 150 kDa, while the Mw of antigen is appr. 51 kD, therefore it
is estimated that app. 3.3 mAb molecules bind one antigen. With other words, 2.3
adalimumab molecules are inactive towards the antigen. Probably the crosslinking
optimistaion could lead to the improvements in this field.
Monolitic columns with immobilizied
commercial mAb – proving selectivity
The samples were loaded as follows:
1. 1. Blank column; Flow-through
2. Blank column; elution
3. mAb column 5; Flow-through
4. mAb column 5; Elution
5. Aldehyde + mAb column buffer 2; FT
6. Aldehyde + mAb column buffer 2; E
7. Aldehyde + mAb column buffer 1; FT
8. Aldehyde + mAb column buffer 1; E
9. Aldehyde + mAb column buffer 1; E
10. lane marker
11. antigene diluted sample
12. column loading mixture
TNFalpha is trimeric protein with the total mass of 51 kDa. All three subunits are
same, therefore each subunit is 17 kDa, what is clearly seen from the SDS-PAGE.
All low-through samples (except the blank) contained no TNFalpha, but all the
other proteins. The pure TNFalpha is present in the elution fraction as a single
band at 17 kDa, what confirmed the column performance.
Monoclonal antibody: anti-Transferrin
o Approx. 10 mg of an anti-transferin from Medri (production and
purification in US).
o from MEDRI (email in Sept13):“We have made a product
analysis on the BioxCell purified Ab and we are not satisfied with
the result (attach). As you can see, there are many degradationproducts and they can not confirm that this is not due to their
fault. “
Monoclonal antibody: anti-Transferrin
- purification on protein A monolithic columnIn order to remove the contaminants we loaded the sample on pA
monolith. The Flow-through and elution fractions (see Figure) were
collected and analysed on SDS PAGE. Obviously (see rows 2, 3 and 5) the
pA does not bind selectively anti-transferrin.
1
ladder
2
Anti-Transferrin FT
3
Anti-Transferrin wash
4
Anti-Transferrin EL
5
Anti-Transferrin load
Monoclonal antibody: anti-Transferrin
- purification on protein G monolithic columnAnalysis of binding anti-transferrin to protein G column were performed. The
flow through and elution fractions were collected and analysed by SDS Page
(Figure) with silver staining.
1 ladder
2 Protein G Column - FT
3 Protein G Column - EL
The antibodies were removed from the sample,
because in FT fraction there are no light and heavy
chain bands. The elution contained a band, which
could be assigned for heavy chain and a weak
band, which could be assigned for light chains.
There is still one impurity seen at app. 100 kDa,
which could be a consequence of not enough
reduced IgG molecules. Regarding the result, we
decided to proceed with purification of higher
amount of anti-transferrin on a protein G disk in
the following step.
Monoclonal antibody: anti- Fibrinogen
o Aprrox. 7 mg of an anti-fibrinogen-mAb (2 types of mAb due to
2 different clones, from BF Ljubljana
o In order to remove the contaminants we loaded the
sample on pA monolith.
MAB @ fibrinogen 1H5/B7
1&2: Load - unreduced and reduced
3&4: CIMAC pA FT- unreduced and reduced
5&6: CIMAC pA E - - unreduced and reduced
Issues
o Further imobilization of mAb just on pA or pG crosslinked and
hydrazide monolithic columns?
o How to receve more pure monoclonal antibody? – purification
in BIASep
o Buy a polyclonal anti-transferin and check the imobilization
procedure?