Antibody affinity purification resin solutions

26 Jun 2025 by AntBio

1. Background

◆ Protein A is a surface protein in the cell wall of Staphylococcus aureus, which is composed of five homologous Ig-binding domains folded into triple helix bundles;

◆ Protein G is a protein in the cell wall of G and C streptococcus;

◆ Protein A/G is a recombinant protein A protein G fusion protein, which fuses 5 Fc binding domains from protein A and 3 Fc binding domains from protein G.

◆ Protein L is derived from Streptococcus macropepatia and binds to immunoglobulins through L-chain interaction.

Fig.1 Region of action of antibody and antibody affinity ligand

Table 1 Antibody affinity differences of proteins A, G, and L

	Protein A	Protein G	Protein A/G	Protein L
merit	The binding site is in the Fc region between the CH2 and CH3 domains, and the heavy chain within the Fab region of the human VH3 family	It can specifically bind to the Fc segment of the immunoglobulin IgG molecule without affecting the ability of the Fab segment to bind to the antigen molecule	It can bind to all human IgG isoforms, IgA, IgE, and IgM. Protein A/G reduces pH dependence and allows binding at pH 5-8	Binds only to the kappa light chain of the antibody without affecting the antigen binding site of the antibody. It can bind to a wider range of antibody classes, such as human IgG, IgM, IgA, IgE, IgD, as well as antibody fragments such as Fab and scFv containing kappa light chains (human type 1, 3, 4, mouse type 1).
shortcoming			Cannot bind to mouse IgA, IgM, and serum albumin	Does not bind bovine, goat, or sheep derived immunoglobulins
apply	The field of purification, fixation, or detection of immunoglobulins	Isolation and purification or removal of IgG from serum, purification of monoclonal antibodies, isolation of antigen-antibody complexes, etc	Extraction and detection of murine IgG monoclonal antibodies	Purification of monoclonal antibodies from culture medium supplemented with BSA or FCS is also widely used for purification and isolation of genetically modified engineered antibodies

Table 2 Affinity between antibody affinity ligands and antibodies from different sources and subtypes

class	Subtype	Protein A	Protein G	Protein A/G	Protein L
person	IgG1	+++	+++	+++	+++
	IgG2	+++	+++	+++	+++
	IgG3	+	+++	+++	+++
	IgG4	+++	+++	+++	+++
	IgM	+	-	+	+++
	IgD	-	-	-	+++
	IgA	+	-	+	+++
	Fab	+	+	+	+++
Mice	IgG1	+	++	++	+++
	IgG2a	+++	+++	+++	+++
	IgG2b	+++	+++	+++	+++
	IgG3	+++	+++	+++	+++
	IgM	-	-	-	+++
Rats	IgG1	+	++	++	+++
	IgG2a	-	+++	+++	+++
	IgG2b	-	+	+	+++
	IgG2c	+++	+++	+++	+++
cattle	IgG1	+	+++	+++	-
cattle	IgG2	+++	+++	+++	-
goat	IgG1	+	+++	+++	-
goat	IgG2	+++	+++	+++	-
horse	IgG(ab)	+	-	+	NA
	IgG(c)	+	-	+	NA
	IgG(T)	-	+++	+++	NA
rabbit	IgG	+++	+++	+++	+
pig	IgG	+++	+	+++	+++
dog	IgG	+++	+	+++	NA
chicken	IgY	-	-	-	-

Note:- Indicates that it is not included; + indicates the winning combination; +++ indicates the wrong combination; NA means N/A or data is not available.

2. Parameters of antibody affinity purification resin

category	Recombinant Protein A agarose gel FF	Recombinant Protein G agarose gel FF	Heavy basophosphate protein A agarose gel FF	Protein A/G agarose gel FF	Recombinant Protein L agarose gel FF
matrix	4% cross-linked agarose	4% cross-linked agarose	4% cross-linked agarose	4% cross-linked agarose	4% cross-linked agarose
Ligands	Recombinant Staphylococcus aureus protein A, ≥ 6 mg/mL	Recombinant streptococcal protein G, ≥ 6 mg/mL, single-point conjugation	Recombinant alkali-tolerant protein A, ≥ 6mg/mL	Protein A protein G fusion protein, ≥ 6 mg/mL	Recombinant macrodigested streptococcal protein L, ≥6 mg/mL
Particle size range^a	45-165μm
Average particle size	~90μm
Dynamic binding capacity^b	≥40mg h-IgG/mL	≥40mg h-IgG/mL	≥45mg h-IgG/mL	≥40mg h-IgG/mL	≥ 40 mg k light chain h-IgG/mL
Recommended workflow speed^c	60-300cm/h	60-300cm/h	60-300cm/h	60-150cm/h	60-300cm/h
Maximum flow rate and pressure^d	＞900cm/h
Use pH	3-10 (recommended working pH), 3-12 (short-term stable)	3-9 (recommended working pH), 2-10 (short-term stable)	3-10 (recommended working pH), 3-12 (short-term stable)	3-10 (recommended working pH), 3-12 (short-term stable)	2-9 (recommended working pH), 15 mM NaOH (CIP)
Chemical stability	Stable in the following solutions: commonly used aqueous phase buffer, 10-100 mmol/L NaOH, 6 mol/L guanidine hydrochloride, 70% ethanol.	Stable in the following solutions: commonly used aqueous buffer, 6mol/L guanidine hydrochloride, 70% ethanol.	Stable in the following solutions: commonly used aqueous phase buffer, 0.1-0.5mol/L sodium hydroxide, 6 mol/L guanidine hydrochloride, 70% ethanol, etc.	Stable in the following solutions: commonly used aqueous buffer, 0.1-0.5mol/L sodium hydroxide, 6mol/L guanidine hydrochloride, 70% ethanol, etc.	Stable in the following solutions: commonly used aqueous buffer, 6mol/L guanidine hydrochloride, 70% ethanol.

Concentrate:

a: Microspheres with more than 90% volume in this particle size range;

b: DBC10% test conditions are: 10% flow-through, 6min residence time;

c: The recommended flow rate means that the flow rate can meet the use conditions of 2-6min residence time under most column heights, and it is not only within the flow rate range;

D: Maximum test flow rate at 10 cm column height.

3. Application Data

Fig.2 Process solvent stability

Fig.3 Tolerance of different protein A resins to NaOH

Fig.4 Elution curves of different protein G resins, the sample is H-IgG

Fig.5. Resin: Heavy alkali-resistant protein A agarose gel FF (medium-pressure prepacked column) (Cat: abs91003-M);

Sample: Bovine serum, approx. 20 mg IgG/mL resin relative to resin;

Loading and binding buffer: 20mM PB, 150mM NaCl, pH7.4, 10CV;

Elution buffer: 100 mM glycine, pH 3.0, 14 CV;

Clean-in-place (CIP): 100 mM NaOH, 10 CV

Fourth, the product information of Absin antibody affinity purification resin

Catalog number	Product name	specification
abs91001-F	Recombinant Protein A agarose gel FF	10mL/100mL
abs91001-G	Recombinant Protein A Agarose Gel FF (Gravity Prepacked Column)	1mL/5mL
abs91001-M	Recombinant Protein A Agarose Gel FF (Medium Pressure Prepacked Column)	1mL/5mL
abs91002-F	Recombinant Protein G agarose gel FF	10mL/100mL
abs91002-G	Recombinant Protein G Agarose Gel FF (Gravity Prepacked Column)	1mL/5mL
abs91002-M	Recombinant Protein G Agarose Gel FF (Medium Pressure Prepacked Columns)	1mL/5mL
abs91003-F	Heavy basophosphate protein A agarose gel FF	10mL/100mL
abs91003-M	Heavy Base-Resistant Protein A Agarose Gel FF (Medium Pressure Prepacked Column)	1mL/5mL
abs91003-G	Heavy basophosphate protein A agarose gel FF (gravity prepacked columns)	1mL/5mL
abs91004-F	Protein A/G agarose gel FF	10mL/100mL
abs91004-M	Protein A/G Agarose Gel FF (Medium Pressure Prepacked Columns)	1mL/5mL
abs91004-G	Protein A/G agarose gel FF (gravity prepacked columns)	1mL/5mL
abs91005-F	Easy to elute Protein G agarose gel FF	10mL/100mL
abs91005-M	Easy Eluting Protein G Agarose Gel FF (Medium Pressure Prepacked Columns)	1mL/5mL
abs91005-G	Easy Eluting Protein G Agarose Gel FF (Gravity Prepacked Column)	1mL/5mL
abs91006-F	Recombinant Protein L agarose gel FF	10mL/100mL
abs91006-M	Recombinant Protein L Agarose Gel FF (Medium Pressure Prepacked Column)	1mL/5mL
abs91006-G	Recombinant Protein L Agarose Gel FF (Gravity Prepacked Column)	1mL/5mL

References

[1] Arora S, Saxena V, Ayyar BV. Affinity chromatography: A versatile technique for antibody purification. Methods. 2017 Mar 1; 116:84-94. doi: 10.1016/j.ymeth.2016.12.010. Epub 2016 Dec 22. PMID: 28012937.

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