Pepsin Actigel

Papain is a nonspecific cysteine protease, which cleaves IgG at the hinge region generating three fragments of ~ 50kDa (two Fabs and one Fc fragment). Antibodies digested with Papain enzyme cannot carry out agglutination, opsonization, precipitation, or lysis, but the Fab fragments are active and would bind and neutralize the respective antigens. Coupling of Papain with the Superflow resin increases the stability of papain enzyme, facilitates controlled digestion, and avoids papain contamination in the digested products. The Fabs can be further purified on the Protein A-Superflow column to get rid of the Fc fragments, and the flow-through will have pure and active Fabs.

Product Code: 6701
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Table 1. Resin Characteristics

Bead Material Agarose
Bead Percentage 4%
Bead Size60-160 µm
Flow Rate1>15 mL/min (>850 cm/hr) @ 25oC
Reactive Group Concentration >3 mg/mL
pH Stability22-7
Storage Temperature2-8o C
Storage Buffer 0.1M sodium acetate, 0.05% sodium azide, 50% glycerol, pH = 4.5, 2-8oC
FormSlurry
Chemical Stability2Stable in all commonly used aqueous solutions and buffers.
Physical Stability2Negligible volume variation due to changes in pH or ionic strength.

1Linear flow rate = volumetric flow rate (cm3/h)/column cross-sectional area (cm2)

2Data refer to the coupled product, provided that the ligand can withstand the pH or chemical environment. Please note the following: pH stability, long term refers to the pH interval where the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pH stability, short term refers to the pH interval for regeneration and cleaning procedures.

Instructions for Use

Pepsin Actigel was developed for the production of F(ab’)2 fragments from IgG molecules. It is intended to be used with Sterogene Bioseparations’ Protein A media. Pepsin has an optimum pH range between 6 and 7 and requires 20 mM Cysteine for activation.

Buffers:
Digestion Buffer: 20 mM Acetate, pH 4.5
Pepsin Actigel Storage Buffer: 0.1M Acetate, 50% Glycerol, 0.05% Azide, pH 4.5
Pepsin Actigel Equilibration Buffer: 20mM Tris, pH 8.5
Pepsin Elution Buffer: 0.1M Glycine, pH2.8 or 0.1M Citrate, pH 2.7
Neutralization Buffer: 1M Tris base
Pepsin Storage Buffer: 20% Ethanol

Protocol:
1. For every 20 mg of IgG use 1 mL Pepsin Actigel in 3 mL Digestion Buffer (optimal IgG concentration is 5 mg/mL in Digestion Buffer + resin).
2. Optimize digestion time, between 2-18 hours; 6 hours is typical for IgG at 5 mg/mL at 37oC.
3. Remove supernatant and adjust pH to 8.5 with Tris base.
4. Regenerate Pepsin Actigel with 10 volumes Digestion and Regeneration Buffer.
5. Load on Protein A medium and equilibrated with 20 mM Tris, pH 8.5.
6. Collect fractions and assay for F(ab’)2 – 3 bed volumes should be sufficient.
7. Store F(ab’)2 fragments under conditions optimized for the specific fragments.
8. Elute bound material from Protein A medium until the absorbance reaches baseline.
9. Re-equilibrate Protein A medium in Equilibration Buffer until pH of eluent is 8-8.5.
10. Store Pepsin Actigel and Protein A medium in the appropriate storage buffer above.

To Download Instructions for use:
INST 6701 Pepsin Actigel

Safety Data Sheets

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