Antibody purification method

Abstract

Disclosed are methods by which compounds/molecules capable of binding antigens, for example antibody type compounds/molecules, can be purified, extracted and/or selected. The methods may be used to purify, extract or select a specific type (or types) of binding agent from a mixed composition. The methods may be used to extract or purify specific binding agents from mixed compositions, which compositions comprise other agents capable of binding other antigens. The methods may find particular application as methods for the purification of blood group antigen antibodies.

Claims

1. A method of purifying an antibody with specificity for a certain or specific blood group antigen, said method comprising: subjecting a red blood cell or red blood cells to an antigen blocking or neutralising process or protocol prior to use, wherein the antigen blocking or neutralising process or protocol blocks or neutralises only those antigens which may bind antibodies which are not to be purified; contacting blood, a blood product, serum and/or plasma with the red blood cell or red blood cells under conditions which permit binding between unblocked/not neutralised blood group antigen(s) and antibody within the blood, blood product, serum and/or plasma; and isolating the bound antibody, wherein the method does not exploit cell membrane fragments or stroma.

2. The method of claim 1, wherein the method uses one or more red blood cells expressing one or more blood group antigens.

3. The method of claim 1, wherein the blood, blood product, plasma and/or serum is derived from or provided by a subject intending to donate blood (blood donors) and/or a subject immunised so as to produce antibodies to a specific target.

4. The method of claim 1, wherein the antibody is a polyclonal antibody.

5. The method of claim 1, wherein the red blood cell or red blood cells are not conjugated to a support, support substrate or polymer.

6. The method of claim 1 wherein the step of isolating the bound antibody involves dissociating and/or eluting antibodies absorbed or bound to the surfaces of the red blood cell(s).

7. The method of claim 6, wherein the dissociating and/or eluting procedures include the use of: heat, rapid freeze/thaw, ultrasonic baths, cell lysis, acid based elution/dissociation, cold acid, organic solvents, xylene, chloroform, modified heat/agitation or chloroquine diphosphate.

8. The method of claim 6, wherein the step of isolating the bound antibody involves contacting antibody/cell complexes with an elution buffer.

9. The method of claim 8, wherein the elution buffer is formulated to bring about antibody dissociation through acid elution.

10. The method of claim 8, wherein the elution buffer comprises, consists essentially of or consists of Glycine-HC1 and NaCl.

11. The method of claim 1, wherein the antigen blocking or neutralising process or protocol uses antibodies as blocking or neutralising agents.

Description

(1) The present invention will now be described in detail with reference to the following figures which show:

(2) FIG. 1: Source plate layout for slide printing.

(3) FIG. 2: Schematic of spot placement per mini-array.

(4) FIG. 3: Polyclonal Anti-D and Polyclonal K Affinity Purification Run

(5) FIG. 4: Image from Assay Results, Slide 1-4 (sample diluent: 4% Dextran, 4% PEG in 2% BSA/PBS)

(6) FIG. 5: Results from all wells of assay

(7) FIG. 6: An example method for the purification of polyclonal antibodies.

EXPERIMENTAL DATA

Introduction

(8) Not all red blood cell (RBC) group antigens (including those required to be tested by the MosaiQ™ instrument) have the corresponding antibody from expressing cell lines. Therefore purification of polyclonal antibodies is required. Current methods of purification are is not sufficient since in plasma (including human plasma) there are many different antibody classes present, including multiple IgG isoforms specific to many different antigens. An efficient method for the purification of antibodies, especially blood group antibodies and in particular polyclonal antibodies, is required.

(9) The aim of the experiment detailed in this example is to purify polyclonal antibodies from patient plasma by a method which exploits adsorption techniques, a low pH elution procedure and (IgG) affinity purification.

(10) This large scale experiment used 25 mL of polyclonal plasma. On-slide specificity of purified fractions, utilising the current manual antigen typing assay was also evaluated.

Materials and Methods

(11) Polyclonal Adsorption and Low pH Elution Procedure

(12) Plasma containing Anti-D was incubated at a ratio of 2:1 (50 mL Plasma:25 mL Cells) with RhD Positive red cells at a 50% haematocrit for 1 hour at 37° C. The same procedure was carried out for plasma containing Anti-K and Anti-Fy.sup.a with corresponding red cells. It should be noted that cells at higher haematocrit, for example about 55%, about 60%, about 65%, about 70%, about 75% or about 80% may be utilised. Further alternate ratios of plasma to cells may be used. For example the ratio of plasma to cells may be about 3:1 or about 4:1. Where the haematocrit of the red blood cells is higher than about 50%, the ratio of plasma to cells may be higher than about 2:1.

(13) After adsorption, elution of the antibodies bound to the red cells was carried out. In order to extract haemolytic cells prior to incubation, the red cell solutions were washed six times with Saline (3000 rpm, 5 min, acceleration 9, deceleration 3. Cells were then incubated at a volume of 2:1 (50 mL elution buffer: 25 mL cells) at room temperature for 2 minutes. A volume (10% of cell mixture volume) of Neutralisation Buffer+154 mM% NaCl was added mixed and centrifuged at 2000×g for 60 seconds. The supernatant was removed and transferred to a clean test tube. The eluate was concentrated to 5 mL and diluted 1:10 in Protein G Binding Buffer in preparation for protein G affinity purification.
Acceptance Criteria: Prior to starting red blood cells may be tested for potency and specificity. Red cell potency criteria should react with homozygote and heterozygote cells (in other words, the cell is selected such that irrespective of the level of antigen expression (strong (homozygote) or weak (heterozygote)), it is still capable of binding antibodies) Cell(s) should be selected to express the antigen or antigen(s) of interest. A cell for use may have a limited or restricted specificity—i.e. it may express the antigen(s) of interest but lack other antigens of no interest. red blood cells may have a 50% haematocrit tested by a validated analyser or specified in blood donation. Ideally, cells should be <4 weeks old.
ÄKTA Purification

(14) The ÄKTA Pure method queued three affinity purifications in sequence through two IgG Protein G HiTrap columns in positions 1, 2 and 3. The method included sequential equilibration and up-flow elution of the column. Samples were injected through the sample loop. Flow rate was lowered to 0.1 mL/min during sample loading.

(15) TABLE-US-00001 TABLE 4 Inlet for affinity purification Inlet Pump Buffer A1 Protein G Binding Buffer A2 Protein G Elution Buffer A3 Elix H.sub.2O

(16) The Protein G column (column position 1, 2 and 3) was equilibrated with 5 mL ELIX H.sub.2O, followed by 25 mL Protein G Binding Buffer before use. The general run method is shown in the Table 5.

(17) TABLE-US-00002 TABLE 5 Protocol specifics for affinity purification Process Buffer Steps Volume (mL) Column equilibration 30 (5 mL H.sub.2O + 25 mL Binding Buffer) Binding Buffer 5 Sample supernatant 1 Binding buffer 20 Elution buffer 25 Binding buffer 5

(18) Once the peak was detected, fractions were collected in 0.5 mL volumes into 96 deep well plates containing 50 μL of Neutralisation Buffer.

(19) Buffer exchange was required immediately after purification due to the low pH of the elution buffer (0.1M Glycine pH 2-3); this was carried out using a Zeba desalting column. The peak fraction was identified through ÄKTA UV trace and total protein levels.

(20) Acceptance Criteria

(21) A constant flow rate of 1 ml/min.sup.−1 and pressure <0.5 MPa was maintained throughout the affinity purification process. The standard sampling loading/column washing/elution phases were clearly defined in the UV chromatogram of the AC process.
Concentration of Samples

(22) Following affinity purification, the 1 mL IgG fractions from each specificity were pooled and concentrated by Ultracel centrifugal filters to a volume of 200 μL, ×5 concentrated (method as per IFU).

(23) Fractions Printed Onto Array

(24) TABLE-US-00003 TABLE 6 Concentration of probes printed onto antigen typing arrays. Serial Dilution Starting Dilution (μL) 50% Reagent Probe Conc. (mg/mL) Ab PBS Glyc Stock Glyc/PBS Polyclonal Anti 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 20 0 20 20 20 D Polyclonal Anti 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 20 0 20 20 20 K Polyclonal Anti- 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 20 0 20 20 20 Fy.sup.a ESD1 (anti-D) 1/10, 1/20, 1/40, 1/80, 1/160, 10 40 50 50 50 1/320 MS56 (anti-K) 1/8, 1/16, 1/32, 1/64, 1/128, 12.5 37.5 50 50 50 1/256 DG-FYA-02 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 20 0 20 20 20 (anti-Fy.sup.a) Anti- 1/10, 1/20, 1/40, 1/80, 1/160, 10 40 50 50 50 Gylcophorin 1/320

(25) Following affinity purification, the purified polyclonal antibodies were printed onto epoxysilane slides at the concentrations shown in Table 6. Anti-D (ESD1 V140550 B01), Anti-Fy.sup.a (DG-FYA-02, J25541 B01) and Anti-K (MS56 J25922 B01) were included as assay controls and to aid placement of grids for image analysis.

(26) Slides were printed with the antibodies listed in Table 6 in a 16 mini array format. Each mini array consisted of a 12 by 12 rectangular grid. This was achieved by 8 source-plate visits with 4 drops per spot. In order to identify whether cross talk (between print-head nozzles) and carryover (between source plate visits) occurred during printing of the slides, 50% Glycerol/PBS was printed in alternate rows of the probe grid (FIGS. 1 and 2). A positive response in any of these probe sites would highlight print head malfunction or the printing of antibodies at too high a concentration. To limit carryover five printhead and Jetspyder purges and the incorporation of a wash plate (filled with Arrayjet buffer) were used. Slides were stored in a slide holder at 2-8° C. in the dark for 5 days prior to assay. FIGS. 1 and 2 show the source plate layout and resulting print layout (spot placement).

(27) Acceptance Criteria:

(28) Visual check of the slides and spots to check printing was successful.

Example Methods

(29) An example method for the purification of polyclonal antibodies is provided in FIG. 6.

(30) A further exemplary method according to this invention may be summarised as follows:

(31) Prior to processing, donor red cells will be washed 3 times with 0.9% Saline (3500 RPM for 5 min, acc 9, dec 7.) Plasma is incubated with red cells 70% haematocrit)at a ratio of 3:1 (Plasma:Cells) for 1 hour at 37° C. with gentle agitation every 15 mins. The red cell/plasma solution is then centrifuged (3500 RPM for 5 min, acc 9, dec 7.) and the supernatant is removed without disruption of red cell pellet. The supernatant volume is then replaced by saline solution. The solution is then centrifuged (3500 RPM for 5 min, acc 9, dec 7.) The wash protocol is repeated for a total of six rounds. Following the final wash, the supernatant is removed. The packed cells are then mixed with elution buffer at a 2:1 ratio (elution buffer:cells) (Elution buffer=0.1M Glycine-HCl, pH 2.7+154 mM NaCl). This mixture is then incubated at room temperature for 2 minutes. Neutralisation buffer (1M Tris-HCl pH 9.0+154 mM NaCl) is then added representing 5% of the total volume of elution buffer to the cell mixture. This solution is then mixed gently and centrifuged at 3500 RPM for 5 minutes (acc 9, dec 7). The supernatant is then transferred to a clean container and the pH measured and any further adjustments made to make it pH 7. The supernatant is diluted 1:1 in Protein G binding buffer. The Protein G column is equilibrated with binding buffer. The eluted antibody/binding buffer solution is injected onto the chromatography column at a low flow rate, while maintaining a constant pressure. Following sample addition, the column is washed with 10 column volumes of binding buffer to wash-out the unbound antibody or contaminating proteins. 10 column volumes of Protein G elution buffer is then flushed through the column in an upward flowpath in order to elute the bound antibody. The eluted antibody is then dialysed into PBS and concentrated. The eluted antibody is collected for characterisation (gels and serology). The concentrated antibody is then diluted in PBS+50% glycerol and printed to an epoxysilane surface. Binding is then investigated using the manual antigen typing assay and with positive and negative cells.