Purification Using Protein A-based Chromatography Media

What is Protein A?

Protein A is derived from a strain of Staphylococcus aureus and contains five regions that bind to the Fc region of IgG. As an affinity ligand, protein A is coupled to Sepharose^® so that these regions are free to bind. One molecule of coupled protein A can bind at least two molecules of IgG.

Both native protein A (nProtein A) and recombinant protein A (rProtein A) ligands are available from Cytiva. These molecules share similar specificity for the Fc region of IgG, but the recombinant protein A has been engineered to include a C-terminal cysteine that enables a single-point coupling when the protein is coupled to Sepharose, which ensures higher binding capacity. Besides the well-known affinity for the Fc region of IgG, protein A also has affinity for certain variants of the Fab region, and consequently, protein A chromatography media can in some cases be used for the purification of Fab and F(ab´)2 fragments. Protein A Sepharose^® chromatography media from Cytiva also possess a considerably higher binding capacity than Protein G Sepharose^® chromatography media and therefore the preferred choice for capture of monoclonal antibodies in industrial-scale processes (see Chapter 7). nProtein A Sepharose^® 4 Fast Flow is manufactured without using animal-derived components. rProtein A is produced in E. coli and no human IgG affinity step is used during validated fermentation and purification processes, minimizing risk of human IgG contamination.

Protein A Sepharose^® High Performance chromatography medium provides sharper eluted peaks and more concentrated elution of antibodies compared with Protein A Sepharose^® 4 Fast Flow. However, the smaller bead size of the Sepharose^® High Performance compared with that of the Sepharose^® 4 Fast Flow matrix leads to increased back pressure on the column. The larger bead size of Sepharose^® 4 Fast Flow allows higher flow rate, which is essential when scaling up a purification.

Protein A vs. Protein G Media

Protein A chromatography media are often a better choice than protein G for isolating certain subclasses of IgG or for removing, for example, cross-species IgG contaminants from horse or fetal calf serum. Although IgG is the major human immunoglobulin, some other types have also been demonstrated to bind with protein A (see IgA and IgM in section Purification of other classes of antibodies later in this chapter).

The binding strength of protein A to IgG depends upon the source species of the immunoglobulin. The dynamic binding capacity depends upon the binding strength and factors such as flow rate during sample application.

Leakage of ligands from an affinity chromatography medium must be considered, especially if harsh elution conditions are used. The multipoint attachment of protein A to Sepharose^® results in very low ligand leakage over a wide range of elution conditions. Removal of ligand contaminant can be achieved by polishing using SEC or IEX.

The various purification options for Protein A Sepharose^® chromatography media are summarized in Table 3.4. Table 3.5 describes typical binding and elution conditions for Protein A Sepharose^® chromatography media.

Table 3.4 Purification options for IgG using protein A Sepharose® chromatography media

Product	Format or column size	Binding capacity (mg IgG/mL medium)	Description
nProtein A Sepharose® 4 Fast Flow	5 mL 25 mL	Approx. 30 (human)	Supplied as suspension for packing in, e.g., XK and Tricorn columns; used for scaling up purification.
Protein A HP MultiTrap™	96-well plates	Approx. 20 (human)	For small-scale purification of IgG, screening of different protein constructs, optimization of buffer conditions, protein enrichment by immunoprecipitation.
Protein A HP SpinTrap™	100 μl Spin columns	Approx. 20 (human)	For small-scale purification of IgG and fragments, protein enrichment by immunoprecipitation.
HiTrap™ Protein A HP	1 mL, 5 mL columns	Approx. 20 (human)	Laboratory-scale purification of IgG and fragments. Prepacked 1 mL or 5 mL columns can be connected in series to facilitate scale-up.
rProtein A Sepharose® Fast Flow	5 mL 25 mL	Approx. 50 (human)	Supplied as suspension for packing in, e.g., XK and Tricorn columns; used for scaling up purification. Higher binding capacity than nProtein A Sepharose® 4 Fast Flow.
rProtein A GraviTrap™	1 mL Gravity columns	Approx. 50 (human)	Fast and efficient manual purification of monoclonal and polyclonal antibodies.
rProtein A/ Protein G GraviTrap™	1 mL Gravity columns	Approx. 35 mg IgG/column (human)	Fast and efficient manual purification of monoclonal and polyclonal antibodies.
HiTrap™ rProtein A FF	1 mL, 5 mL columns	Approx. 50 (human)	Laboratory-scale purification of IgG, fragments, and subclasses. Prepacked 1 mL or 5 mL columns can be connected in series to scale up. Enhanced binding capacity.
Ab Buffer Kit	1 Kit		Premade buffers recommended for antibody purification using Protein A HP MultiTrap™, Protein A HP SpinTrap™, HiTrap™ Protein A HP, and HiTrap™ rProtein A FF.

Table 3.5 Binding and elution conditions commonly used with Protein A Sepharose® chromatography media for purification of IgG from different species.

Species	Subclass	Protein A binding pH	Protein A elution pH
Human	IgG1 IgG2 IgG3 IgG4	6.0 to 7.0 6.0 to 7.0 8.0 to 9.0 7.0 to 8.0	3.5 to 4.5 3.5 to 4.5 ≤ 7.0 3.0 to 6.0
Cow	IgG2	n.a.	2.0
Goat	IgG2	n.a.	5.8
Guinea pig	IgG1 IgG2	n.a. n.a.	4.8 4.3
Mouse	IgG1 IgG2a IgG2b IgG3	8.0 to 9.0 7.0 to 8.0 Approx. 7.0 Approx. 7.0	4.5 to 6.0 3.5 to 5.5 3.0 to 4.0 3.5 to 5.5
Rat	IgG1 IgG2a IgG2b IgG3	≥ 9.0 ≥ 9.0 ≥ 9.0 8.0 to 9.0	7.0 to 8.0 ≤ 8.0 ≤ 8.0 3.0 to 4.0 (using 3 M potassium isothiocyanate)

Notes on Use

Binding strengths are tested with free protein A and can be used as guidelines to predict the binding behavior to a protein A purification medium. However, when coupled to an affinity matrix, the interaction can be altered. For example, rat IgG₁ does not bind to protein A, but does bind to Protein A Sepharose^®.

With some antibodies, for example mouse IgG₁, a high concentration of sodium chloride in the binding buffer might be necessary to achieve efficient binding. Recommended binding buffers are 1.5 M glycine, 3 M sodium chloride, pH 8.9 or 0.02 M sodium phosphate, 3 M sodium chloride, pH 7.0.

Most antibodies and subclasses bind protein A close to physiological pH and ionic strength. Avoid excessive washing if the interaction between the protein of interest and the ligand is weak since this might decrease yield.

Use a mild elution method when labile antibodies are isolated. Reverse the flow of the wash buffer and elute with 0.1 M glycyltyrosine in 2 M sodium chloride, pH 7.0 at room temperature, applied in pulses. (Note: glycyltyrosine absorbs strongly at wavelengths used for detecting proteins). The specific elution is so mild that the purified IgG is unlikely to be denatured. Alternative elution buffers include: 1 M acetic acid pH 3.0, 0.1 M glycine-HCl pH 3.0, or 3 M potassium isothiocyanate. Note: potassium isothiocyanate can severely affect structure and immunological activity.

Desalt and/or transfer purified IgG fractions into a suitable buffer using a desalting column.

To increase capacity, connect several HiTrap columns (1 mL or 5 mL) in series. Alternatively pack a larger column with nProtein A Sepharose^® 4 Fast Flow or rProtein A Sepharose^® 4 Fast Flow (see Column packing and preparation). When working with large-scale fermentation, consider using any of the MabSelect chromatography media. MabSelect is designed to retain a high binding capacity at the higher flow rates required to process large sample volumes as rapidly as possible (see MabSelect chromatography media and prepacked columns later in this chapter).

Reuse of Protein A Sepharose^® chromatography media depends on the nature of the sample and should only be considered when processing identical samples to avoid cross-contamination.