Antibody classes Characteristic lgM Heavy chain Light chair c or A Ystructure Fig.10.Antibody classes IgG,IgG fragments and subclasses The basis for purification of IgG,IgG fragments and subclasses is the high affinity of protein A and protein G for the Fc region of polyclonal and monoclonal IgG-type antibodies,see Figure 9. Protein A and protein G are bacterial proteins (from Staphylococcus aureus and Streptococcus, respectively)which,when coupled to Sephascreare extremely useful casy to use medi rou Pplicatio xamplesinclude the pu urification of monocl nal IgG-typ of olvclonal IgG subclass nd the ads complex g IgG.IgG subclass can be solated fr s fluid.cell ison of the engths of protein A and protein G to A useful reference on this subject is also:Structure of the IgG-binding regions of streptococcal Protein G,EMBO.,5,1567-1575(1986). Binding strengths are tested with free protein A or protein G and can be used as a guide to predict the binding behaviour to a protein A or protein G purification medium.However, when coupled to an affinity matrix,the interaction may be altered.For example,rat IgG does not bind to protein A,but does bind to Protein A Sepharose
26 Fig. 10. Antibody classes. IgG, IgG fragments and subclasses The basis for purification of IgG, IgG fragments and subclasses is the high affinity of protein A and protein G for the Fc region of polyclonal and monoclonal IgG-type antibodies, see Figure 9. Protein A and protein G are bacterial proteins (from Staphylococcus aureus and Streptococcus, respectively) which, when coupled to Sepharose, create extremely useful, easy to use media for many routine applications. Examples include the purification of monoclonal IgG-type antibodies, purification of polyclonal IgG subclasses, and the adsorption and purification of immune complexes involving IgG. IgG subclasses can be isolated from ascites fluid, cell culture supernatants and serum. Table 2 shows a comparison of the relative binding strengths of protein A and protein G to different immunoglobulins compiled from various publications. A useful reference on this subject is also: Structure of the IgG-binding regions of streptococcal Protein G, EMBO J., 5, 1567–1575 (1986). Binding strengths are tested with free protein A or protein G and can be used as a guide to predict the binding behaviour to a protein A or protein G purification medium. However, when coupled to an affinity matrix, the interaction may be altered. For example, rat IgG1 does not bind to protein A, but does bind to Protein A Sepharose. Antibody classes Characteristic IgG k l or k l or k l or k l or k l or g Y structure Light chain Heavy chain IgM IgA IgE IgD m a e d
Table 2.Relative binding strnths of protein A and protinGtovarious immunoglobuinNo binding:- relative strength of binding:+,++,+++,+++ Protein A Species Subclass binding Human variable e8 ++ Sheep ingle step purification based on Fe region specificity will co-purify host IgG and may even bind trace amounts of serum proteins.To avoid trace amounts of contaminating IgG,consider alternative techniques such as immunospecific affinity (using anti-host IgG antibodies as the ligand to remove host IgG or target specific antigen to avoid binding host IgG),ion exchange or hydrophobic interaction chromatography(see Chapter 6). Both protein A and a recombinant protein A are available,with similar specificities for the Fc region of IgG.The recombinant protein A has been engineered to include a C-terminal cysteine that enables a single-point coupling to Sepharose.Single point coupling often results in an enhanced binding capacity. Genetically engineered antibodies and antibody fragments can have altered biological properties and also altered properties to facilitate their purification.For example,tags can be introduced into target molecules for which no affinity media were previously available thus creating a fusion protein that can be effectively purified by affinity chromatography. Details for the purification of tagged proteins are covered in the section Recombinant Fusion Proteins on page 42 of this handbook.For information on the purification of recombinant proteins in general,refer to The Recombinant Protein Handbook:Protein Amplification and Simple Purification and the GST Fusion System Handbook from rsham biosciences 27
27 Table 2. Relative binding strengths of protein A and protein G to various immunoglobulins. No binding: –, relative strength of binding: +, ++, +++, ++++. Protein A Protein G Species Subclass binding binding Human IgA variable – IgD – – IgE IgG1 ++++ ++++ IgG2 ++++ ++++ IgG3 – ++++ IgG4 ++++ ++++ IgM* variable – Avian egg yolk IgY** – – Cow ++ ++++ Dog ++ + Goat – ++ Guinea pig IgG1 ++++ ++ IgG2 ++++ ++ Hamster + ++ Horse ++ ++++ Koala – + Llama – + Monkey (rhesus) ++++ ++++ Mouse IgG1 + ++++ IgG2a ++++ ++++ IgG2b +++ +++ IgG3 ++ +++ IgM* variable – Pig +++ +++ Rabbit no distinction ++++ +++ Rat IgG1 – + IgG2a – ++++ IgG2b – ++ IgG3 + ++ Sheep +/– ++ * Purify using HiTrap IgM Purification HP columns. ** Purify using HiTrap IgY Purification HP columns. Single step purification based on Fc region specificity will co-purify host IgG and may even bind trace amounts of serum proteins. To avoid trace amounts of contaminating IgG, consider alternative techniques such as immunospecific affinity (using anti-host IgG antibodies as the ligand to remove host IgG or target specific antigen to avoid binding host IgG), ion exchange or hydrophobic interaction chromatography (see Chapter 6). Both protein A and a recombinant protein A are available, with similar specificities for the Fc region of IgG. The recombinant protein A has been engineered to include a C-terminal cysteine that enables a single-point coupling to Sepharose. Single point coupling often results in an enhanced binding capacity. Genetically engineered antibodies and antibody fragments can have altered biological properties and also altered properties to facilitate their purification. For example, tags can be introduced into target molecules for which no affinity media were previously available thus creating a fusion protein that can be effectively purified by affinity chromatography. Details for the purification of tagged proteins are covered in the section Recombinant Fusion Proteins on page 42 of this handbook. For information on the purification of recombinant proteins in general, refer to The Recombinant Protein Handbook: Protein Amplification and Simple Purification and the GST Fusion System Handbook from Amersham Biosciences
HiTrap Protein G HP,Protein G Sepharose 4 Fast Flow,MAbTrap Kit Protein G,a cell surface protein from Group G streptococci,is a type IlI Fc-receptor. Protein G binds through a non-immune mechanism.Like protein A,protein G binds specifically to the Fc region of IgG,but it binds more strongly to several polyclonal IgGs (Table 2)and to human IgG.Under standard buffer conditions,protein G binds to all human subclasses and all mouse IgG subclasses,including mouse IgG.Protein G also binds rat IgG2 and IgGab,which either do not bind or bind weakly to protein A. Amersham bios offers a recombinant form of protein G from which the albumin binding region of the nativ e molecule has been deleted etically thereby s with alb min.Recombinant protein G Protein G Sepharose is a better choice for general purpose apture of antibodies since it a broader range of IgG from euka ses of Igc minimal s.The binding strength of protein G for IgG depends on the source species and subclass of the immunoglobulin. The dynamic binding capacity depends on the binding strength and also on several other factors,such as flow rate during sample application. Many antibodies also interact via the Fab region with a low affinity site on protein G Protein G does not appear to bind human myeloma IgM,IgA or IgE,although some do bind weakly to protein A. Leakage of ligandsfrom an affinity medium is always a possibility,especially if harsh elution conditions are used.The multi-point attachment of protein G to Sepharose results in very low leakage levels over a wide range of elution conditions. Purification option Binding capacity Comments HTapG HP MAbTrap Kit Human IgG.25 me/column 4 ml/min ification of lg.fragments and g a rat lgc Human l>20 mg/ml medium 400 cm/h nsion ready Ratl与G.7 ng/ml medium
28 HiTrap Protein G HP, Protein G Sepharose 4 Fast Flow, MAbTrap Kit Protein G, a cell surface protein from Group G streptococci, is a type III Fc-receptor. Protein G binds through a non-immune mechanism. Like protein A, protein G binds specifically to the Fc region of IgG, but it binds more strongly to several polyclonal IgGs (Table 2) and to human IgG3. Under standard buffer conditions, protein G binds to all human subclasses and all mouse IgG subclasses, including mouse IgG1. Protein G also binds rat IgG2a and IgG2b, which either do not bind or bind weakly to protein A. Amersham Biosciences offers a recombinant form of protein G from which the albuminbinding region of the native molecule has been deleted genetically, thereby avoiding undesirable reactions with albumin. Recombinant protein G contains two Fc binding regions. Protein G Sepharose is a better choice for general purpose capture of antibodies since it binds a broader range of IgG from eukaryotic species and binds more classes of IgG. Usually protein G has a greater affinity than protein A for IgG and exhibits minimal binding to albumin, resulting in cleaner preparations and greater yields. The binding strength of protein G for IgG depends on the source species and subclass of the immunoglobulin. The dynamic binding capacity depends on the binding strength and also on several other factors, such as flow rate during sample application. Many antibodies also interact via the Fab region with a low affinity site on protein G. Protein G does not appear to bind human myeloma IgM, IgA or IgE, although some do bind weakly to protein A. Leakage of ligands from an affinity medium is always a possibility, especially if harsh elution conditions are used. The multi-point attachment of protein G to Sepharose results in very low leakage levels over a wide range of elution conditions. Purification options Binding capacity Maximum Comments operating flow HiTrap Human IgG, > 25 mg/column 4 ml/min (1 ml column) Purification of IgG, fragments and Protein G HP Human IgG, >125 mg/column 20 ml/min (5 ml column) subclasses, including human IgG3 . Strong affinity for monoclonal mouse IgG1 and rat IgG. Prepacked columns. MAbTrap Kit Human IgG, > 25 mg/column 4 ml/min Purification of IgG, fragments and subclasses, including human IgG3. Strong affinity for monoclonal mouse IgG1 and rat IgG. Complete kit contains HiTrap Protein G HP (1 x 1 ml), accessories, pre-made buffers for 10 purifications and detailed experimental protocols. Protein G Human IgG, > 20 mg/ml medium 400 cm/h* Supplied as a suspension ready Sepharose 4 Cow IgG, 23 mg/ml medium for column packing. Fast Flow Goat IgG, 19 mg/ml medium Guinea pig IgG, 17 mg/ml medium Mouse IgG, 10 mg/ml medium Rat IgG, 7 mg/ml medium *See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm
Purification examples Figure 11 shows the purification of mouse monoclonal IgG on HiTrap Protein G HP 1 ml. The monoclonal antibody was purified from a hybridoma cell culture supernatant. Immunodiffusion SDS PAGE 25 .non-r Lane 1 2 3 4 510162025 30 Fig.11.Purification of monoclonal mouse IgGon HiTrap Protein GHP,1ml. Figure 12 shows the purification of recombinant mouse Fab fragments,expressed in E.coli,using Protein G Sepharose 4 Fast Flow.Chimeric,non-immunogenic "humanized" mouse Fab,Fab'and F(ab',fragments are of great interest in tumour therapy since they penetrate tumours more rapidly and are also cleared from the circulation more rapidly than full size antibodies. 3.5 Medium: 25 15 inding cetate 0.5 M acetic acid,pH 2.5 200 07 of recombin t fab fragments directed to the sn clope protein gp120 of HIV-1 (anti-gp120 Fab). 29
29 Purification examples Figure 11 shows the purification of mouse monoclonal IgG1 on HiTrap Protein G HP 1 ml. The monoclonal antibody was purified from a hybridoma cell culture supernatant. Sample: 12 ml mouse IgG1 hybridoma cell culture supernatant Column: HiTrap Protein G HP, 1 ml Flow: 1.0 ml/min Binding buffer: 20 mM sodium phosphate, pH 7.0 Elution buffer: 0.1 M glycine-HCI, pH 2.7 Electrophoresis: SDS-PAGE, PhastSystem™, PhastGel™ Gradient 10–15, 1 µl sample, silver stained Immunodiffusion: 1% Agarose A in 0.75 M Tris, 0.25 M 5,5-diethylbarbituric acid, 5 mM Ca-lactate, 0.02% sodium azide, pH 8.6 Lane 1. Low Molecular Weight Calibration Kit, reduced Lane 2. Mouse hybridoma cell culture fluid, non-reduced, diluted 1:10 Lane 3. Pool I, unbound material, non-reduced, diluted 1:10 Lane 4. Pool II, purified mouse IgG1, non-reduced, diluted 1:10 Lane 1 2 3 4 Mr SDS PAGE 14 400 20 100 30 000 45 000 66 000 97 000 Immunodiffusion A280 nm 5.0 2.5 0 5 10 15 20 25 30 ml pool I pool II Binding buffer Elution buffer Binding buffer Fig. 11. Purification of monoclonal mouse IgG1 on HiTrap Protein G HP, 1 ml. Figure 12 shows the purification of recombinant mouse Fab fragments, expressed in E. coli, using Protein G Sepharose 4 Fast Flow. Chimeric, non-immunogenic "humanized" mouse Fab, Fab' and F(ab')2 fragments are of great interest in tumour therapy since they penetrate tumours more rapidly and are also cleared from the circulation more rapidly than full size antibodies. Fig. 12. Purification of recombinant Fab fragments directed to the envelope protein gp120 of HIV-1 (anti-gp120 Fab), expressed in E. coli. Sample: Recombinant Fab fragment from E. coli. Medium: Protein G Sepharose 4 Fast Flow (1 ml) Flow: 0.2 ml/min (60 cm/h), or 0.3 ml/min (90 cm/h) Binding buffer: 0.15 M NaCl, 10 mM sodium phosphate, 10 mM EDTA, pH 7.0 Elution buffer: 0.5 M ammonium acetate, pH 3.0 Wash buffer: 1 M acetic acid, pH 2.5 0 0.5 1.5 2.5 3.5 0.0 Elution buffer UV 280 nm Conductivity pH 10.0 20.0 30.0 40.0 ml A280 nm
Performing a separation Column: HiTrap Protein G HP. Recommended flow rates:1 ml/min (1 ml column)or 5 mVmin (5 ml column) Binding buffer .02 Msodium phosphate,pH7.0 Elution buffer: 0.1 Mglycine-HCI,pH2.7 Neutralization buffer: 1 M Tris-HCI.pH 9.0 to remove cells and debris.Filter th ugh a binding buffer either by buffer exchange on a desalting column or by dilution and pH adjustment(see page 133). 1.Equilibrate column with5column volumes of binding butfer. .Apply sample. 4.Elute with 5 column volumes of elution buffer 5.mmediatelyeatewith5-10com volumes of binding buffe IgGs from most species and subclasses bind to protein G at near physiological pH and ionic strength.For the optimum binding conditions for IgG from a particular species,it is worth t literature.Avoid excessive washing if the interaction between the protein and the ligandis weak,sinc this may drease the yield. Most immunoglobulin species do not elute from protein g sepharose until pH 2.7 or less If biological activity of the antibody or antibody fragment is lost due to the low pH Sepharose:the may. Desalt and/or transfer purified IgG fractions to a suitable buffer using a desalting column (see page 133). 业 Reuse of Protein G Sepharose depends on the nature of the sample and should only be considered when processing identical samples to avoid cross-contamination. To increase capacity,connect several HiTrap Protein G HP columns(1 ml or 5 ml)in series HiTrap columns can be used with a syringe,a peristaltic pump or connected to a liquid chromatography system,such as AKTAprime.For greater capacity pack a larger column with Protein G Sepharose 4 Fast Flow(see Appendix 3). 30
30 Performing a separation Column: HiTrap Protein G HP, 1 ml or 5 ml Recommended flow rates: 1 ml/min (1 ml column) or 5 ml/min (5 ml column) Binding buffer: 0.02 M sodium phosphate, pH 7.0 Elution buffer: 0.1 M glycine-HCl, pH 2.7 Neutralization buffer: 1 M Tris-HCl, pH 9.0 Centrifuge samples (10 000 g for 10 minutes) to remove cells and debris. Filter through a 0.45 µm filter. If required, adjust sample conditions to the pH and ionic strength of the binding buffer either by buffer exchange on a desalting column or by dilution and pH adjustment (see page 133). 1. Equilibrate column with 5 column volumes of binding buffer. 2. Apply sample. 3. Wash with 5–10 column volumes of the binding buffer to remove impurities and unbound material. Continue until no protein is detected in the eluent (determined by UV absorbance at 280 nm). 4. Elute with 5 column volumes of elution buffer*. 5. Immediately re-equilibrate with 5–10 column volumes of binding buffer. *Since elution conditions are quite harsh, it is recommended to collect fractions into neutralization buffer (60 µl – 200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral. IgGs from most species and subclasses bind to protein G at near physiological pH and ionic strength. For the optimum binding conditions for IgG from a particular species, it is worth consulting the most recent literature. Avoid excessive washing if the interaction between the protein and the ligand is weak, since this may decrease the yield. Most immunoglobulin species do not elute from Protein G Sepharose until pH 2.7 or less. If biological activity of the antibody or antibody fragment is lost due to the low pH required for elution, try Protein A Sepharose: the elution pH may be less harsh. Desalt and/or transfer purified IgG fractions to a suitable buffer using a desalting column (see page 133). Reuse of Protein G Sepharose depends on the nature of the sample and should only be considered when processing identical samples to avoid cross-contamination. To increase capacity, connect several HiTrap Protein G HP columns (1 ml or 5 ml) in series. HiTrap columns can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system, such as ÄKTAprime. For greater capacity pack a larger column with Protein G Sepharose 4 Fast Flow (see Appendix 3)