Murine IgG1 binds Protein A weakly, so Protein G is the standard capture ligand for most mouse monoclonals — but the format choice is narrower than for Protein A. Packed-bed Protein G resins dominate the lab scale; for higher throughput, MonoCore™ Protein G offers recombinant Protein G on a modified cellulose monolith with a 15 µm channel size and UNF 10/32 connectors, in 1.8 mL and 5.4 mL capsules that run on any standard FPLC system. Because transport through the monolith is convective, capture cycles run at flow rates a packed-bed Protein G resin cannot sustain. This page is about which ligand and which format to choose; the run itself — buffers, volumes, step times, the serum problem and the species table — is in how to purify an antibody from hybridoma supernatant. Binding capacity, with the conditions it was determined under, is stated in the datasheet.
The ligand costs something at the other end of the cycle, and it belongs in the decision: Protein G binds Fc more tightly and over a larger contact area than Protein A, so it releases at a lower pH — vendor protocols centre on about pH 2.7 against pH 3.0 for Protein A. On an acid-sensitive product that half a unit matters, and the neutralisation has to be immediate rather than merely prompt; see why low-pH elution is the wrong tool for sensitive modalities.
Why Protein G, not Protein A
Ligand specificity, not preference, drives this choice.
- Human IgG1, IgG2, IgG4 bind both ligands strongly.
- Human IgG3 binds Protein A weakly and Protein G strongly.
- Mouse IgG1 — the decisive case — binds Protein A weakly and Protein G strongly.
- Mouse IgG2a and IgG2b bind both ligands well; mouse IgG3 is rated lower on Protein A by several sources, so verify if your line is IgG3.
- Rat IgG binds Protein A weakly and Protein G better — with the exception of rat IgG2b, where neither Fc ligand is convincing and a kappa-specific Protein L is the honest answer, even though we do not sell one. Mouse IgM and IgA bind neither ligand either; a Protein G capsule will not help there.
- Goat, sheep, bovine and equine IgG bind Protein A weakly and Protein G strongly.
- Canine and feline IgG: the vendor tables rank Protein A above Protein G for both species — the mouse rule inverts here, as it does for pig and guinea pig. That rating describes serum IgG, which in dogs is dominated by the IgG-B subclass; published work has captured all four canine subclasses on Protein G resin, so for a monoclonal line of another subclass, screen both rather than trusting either rating.
Two practical consequences follow.
A large share of murine monoclonals from hybridoma lines is IgG1 — exactly the subclass Protein A handles poorly. Running it on Protein A costs yield in the capture step.
One detail worth checking on any product: native Protein G also binds serum albumin. Recombinant Protein G ligands have the albumin-binding domain removed. MonoCore™ Protein G uses recombinant Protein G.
What is available, and where each format fits
- Spin columns and gravity formats cover microgram to milligram amounts. They are the fastest path for a single sample, but they are not a process step.
- Packed-bed Protein G resin covers lab to pilot scale and is well established across suppliers. It is diffusion-limited: flow rate and cycle time are capped by pressure and bed compression.
- Cellulose monolith — MonoCore™ Protein G covers lab to pilot scale in 1.8 mL and 5.4 mL, larger on request. Transport is convective, so cycles are short and the open channel structure tolerates complex feeds. Its limit is the ligand, not the format: Protein G is the right choice where Protein A binds weakly, and the wrong one where Protein A binds well.
- Membrane adsorbers with Protein G are fast and disposable, but the choice of chemistry is limited and process-scale options are few.
The binding capacity of a packed bed collapses as the flow rate rises, because the antibody has to diffuse into the bead pores. In a monolith the mobile phase flows through the channels, so residence time is set by geometry rather than by diffusion. That is why the same capture step can run in minutes instead of an hour.
Where it does not help: if time per cycle is not what limits you, the case for running many short cycles largely disappears, and the format matters less than the chemistry.
MonoCore™ Protein G at a glance
- Ligand: recombinant Protein G
- Matrix: modified cellulose monolith
- Dynamic binding capacity: stated in the product datasheet together with the conditions it was measured under — capacity is feed-specific, so measure it on yours where the conditions differ (how to read a capacity figure).
- Channel size: 15 µm standard; other sizes on request, selected per column
- Format: 1.8 mL · 5.4 mL · larger on request
- Connectors: UNF 10-32 coned port, 1/16″ OD tubing
- Recommended flow: 15 mL/min at 1.8 mL · 45 mL/min at 5.4 mL
- Maximum pressure: 0.8 MPa (8 bar)
- On an affinity capsule the Protein G ligand is the sensitive component and sets the working range. The pH figures quoted for the assembled capsule — 4–13 long term, 2–14 short term — describe the housing and the matrix, not the ligand, and a cleaning regime must not be inferred from them. Conditions come with the datasheet.
Capsules ship pre-assembled and connect to a standard FPLC system, a peristaltic pump or a syringe. Detailed performance data are available on request.
Frequently asked questions
How is a Protein G capsule cleaned?
Within limits, and the limits are the ligand's rather than the matrix's. Protein G is a protein, so alkali tolerance on an affinity capsule is set by the ligand; the concentration and contact time for MonoCore™ Protein G come with the datasheet rather than as a general figure. What is specified publicly, and what that means in practice, is in regeneration, CIP and service life.
Does the flow rate change binding capacity?
In a packed bed it usually does: the target has to diffuse into the bead, and at short residence times it does not get there. Convective transport removes that diffusion limit inside the medium, but film transport and the kinetics of the ligand still apply, so capacity is never fully independent of how fast you run. Determine the working point on your own feed — record a breakthrough curve at two or three residence times (how to read a capacity figure).
What sizes are available above 5.4 mL?
Larger formats are built on request. Talk to us about the volume and the cycle time you need.
Still an open question?
Tell us the molecule, the feed volume and the system you run it on, and we will say whether a MonoCore™ capsule is the right starting point — or whether it is not.