Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a Ryan S. Stowers, 1 Jacqueline A. Callihan, 2 James D. Bryers 2 1 Department of Bioengineering, Clemson University, Clemson, South Carolina 29634 2 Department of Bioengineering, University of Washington, Seattle, Washington 98195 Abstract: Immunoglobulin fragments have advantages over whole antibody molecules in several experimental procedures including immunohistochemistry and in vivo studies. Additionally, IgG fragments have shown great promise for immunotherapy. However, experimental conditions for the production of these Ig fragments are variable, especially in microscale levels. Here we report optimal digestion time, ph, and enzyme to antibody weight ratio for pepsin digestion of mouse monoclonal IgG2a into F(ab )2 antibody fragments. Optimal conditions were determined to be 8 hr with a ph of 4.0 and 1:40 pepsin to antibody weight ratio. 1. INTRODUCTION Immunoglobulin (Ig) fragments can be advantageous for a number of experimental methods and have potential for immunotherapeutic applications in cancer treatment, infection clearance, and targeted drug delivery. F(ab ) 2 fragments are smaller than whole Igs but maintain antigen binding function. The smaller size results in better tissue penetration and less steric hindrance leading to more sensitive antigen detection. Fragments also have reduced non-specific binding and lower immunogenicity in vivo [4]. Immunoglobulins or antibodies are abundant serum proteins that are produced by B cells as an adaptive component of the immune system. Ig molecules are typically composed of two heavy chains and two light chains that are covalently bonded. There are several classes, or isotypes, of Igs based on the properties of the heavy chains. Major heavy chain isotypes are µ, α, δ, ε, and γ corresponding to IgM, IgA, IgD, IgE, and IgG. Each heavy chain isotype can bind with either of the two light chain isotypes; κ and λ [3]. IgG is the most common isotype and is of interest for this study. Its structure is pictured below (Figure 1). IgGs are a class of large proteins of approximately 150 kda, made of two identical heavy chains (50 kda) and two identical light chains (25 kda). Each IgG has two antigen binding sites that can bind a single antigen molecule independently in a process called opsonization [3]. Opsonized particles are then recognized by host phagocytic cells that bind to the Fc portion of antibodies and proceed to engulf and destroy the particle [1]. Fragments of IgG molecules composed of only antigen binding portions can be obtained Figure 1. Model of IgG illustrating heavy and light chain, antigen binding sites, and covalent bonding. Digestion to F(ab ) 2 fragment is also shown.
through enzymatic degradation. The enzyme pepsin cleaves the Fc portion of an Ig into small subfragments leaving a F(ab ) 2 fragment with two antigen binding sites connected by disulfide bonds (Figure 1) [2]. Pepsin is an acidic endopeptidase produced by chief cells in the stomach that functions to degrade proteins into small peptides. It is mostly active at ph 1-2 and is irreversibly denatured above ph 6. For antibody digestion, pepsin immobilized on a resin support is commonly used because the reaction can be stopped by removing the resin and leaves no enzyme in the digest solution. The digestion process is highly variable, resulting in many differing protocols for individual antibody species and amounts digested [5]. For basic research, it is often financially desirable to work with small amounts of monoclonal antibody, on the order of micrograms. However, common protocols for digestion and purification from commercial kits do not produce F(ab ) 2 fragments efficiently. The goal of this study was to provide optimal conditions for digestion of mouse monoclonal IgG2a in microscale. Effects of digestion time, ph, and enzyme-to-antibody weight ratio were investigated. 2. MATERIALS AND METHODS 2.1 Antibody Purification and Concentration IgG2a mouse monoclonal antibodies directed toward the bacterium, Pseudomonas aeruginosa (AbD Serotec), were used as antibodies. Antibodies were shipped in 0.1% bovine serum albumin (BSA) as a preservative. Since BSA is known to affect the digestion rate, the solution was purified on a Protein G NAb Spin Column (Pierce) to remove the antibody from the BSA solution. Recovered IgG was concentrated to 4 mg/ml using an Amicon Ultra 4 ml centrifuge filter. 2.2 Digestion Buffer Digestion buffer was prepared by dissolving 2.72 g sodium acetate, trihydrate in 1L of ultrapure water to prepare a 20 mm solution. The ph was adjusted to 4.0 and the buffer was stored at 4 o C. 2.3 Immobilized Pepsin Immobilized pepsin from Pierce s ImmunoPure F(ab ) 2 Preparation Kit was used for digestion. The vial was swirled to obtain an even suspension and a wide bore pipette tip was used to place the 50% slurry in a 0.6 µl microcentrifuge tube. For time and ph optimizations, 0.4 µl of slurry was added to obtain a pepsin-to-antibody weight ratio of 1:40. For the weight ratio optimization, 1.6 µl and 4 µl of slurry were added to obtain pepsin to antibody weight ratios of 1:10 and 1:4 respectively. Immobilized pepsin was washed with 500 µl digestion buffer, centrifuged, and the pellet was resuspended in digestion buffer for a total volume of 46 µl. 2.4 Digestion 4 µl (16 µg) of prepared IgG was added to the equilibrated immobilized pepsin. The mixture was incubated at 37 o C in a shaking water bath during digestion. Tubes were centrifuged to separate the digest from the immobilized pepsin and 10 µl aliquots of digest were removed for analysis via gel electrophoresis. 2.5 Gel Electrophoresis Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to evaluate the digested antibodies. Digested samples were heated to 95 o C for 5 min. Aliquots were diluted with 20 µl Laemmli Sample buffer. Fifteen µl of diluted sample was added to BioRad 12 lane, 4-20% acrylamide, Ready Gels. Gels were run at 150 V constant, 35 ma max for 60 min in a BioRad Mini Protean II system, stained for Gel Code Blue (Pierce) and analyzed using Image J software to determine protein band intensities. 2.6 Digestion Time Optimization The digestion was carried out for a total of 24 hr and 10 µl aliquots were taken at 1, 2, 3, 4, 5, 6, 8, 12, and 24 hr to compare F(ab ) 2 fragment generation. 2.7 ph Optimization Digestion buffer ph was adjusted to 3.0, 4.0, 4.5, 5.0, and 6.0. The digestion was carried out
for 4 hr and 10 µl aliquots were analyzed from each condition. 2.8 Immobilized Pepsin to Antibody Ratio Digestions were carried out for 4 hr with immobilized pepsin to antibody weight ratios of 1:4, 1:10, 1:40. Ten µl aliquots were taken from each condition for analysis. 3. RESULTS SDS-PAGE gels have two bands of interest in determining digestion of IgG antibody. The band at 160 kda shows whole IgG while the 115 kda band indicates F(ab) 2 fragments (Figure 2). It is clear that digestion is occurring after 5 hr from the steadily decreasing intensity of the whole IgG band and the steadily increasing band of fragments. An 8 hr digestion yields the greatest intensity F(ab ) 2 band without over-digestion, cleavage of F(ab ) 2 into F(ab) or smaller fragments. The optimal ph was determined to be 4.0 when gel bands were analyzed using Image J software and image intensity was graphed (Figure 3). At ph 3.0, immobilized pepsin was Figure 2. (a) SDS-PAGE gel from digestion time optimization study showing 160 kda whole IgG2a band and 115 kda F(ab ) 2 fragment band. (b) Results from gel analysis showing relative band intensity versus digestion time with whole IgG2a (grey) and F(ab ) 2 (black).
over-activated, causing it to digest the IgG molecules to small subfragments. A ph of 6.0 largely inactivated immobilized pepsin, causing it to give very low amounts of F(ab ) 2 fragments. The optimal immobilized pepsin to IgG antibody weight ratio was 1:40. As seen in Figure 4, it was clear that the initial ratio of 1:4 contained too much pepsin for good digestion. 4. DISCUSSION Optimal conditions for immobilized pepsin digestion of monoclonal mouse IgG2a were determined to be 8 hr at ph 4.0 with a 1:40 pepsin to antibody weight ratio. For maximum efficiency, these conditions could be even further refined. A more precise ph could be determined by testing from ph 4.0 to 5.0 in 1/10 th increments. It is difficult to determine the trend in this range from the data collected because relative intensities of both fragments and whole antibody are higher at ph 5.0 than 4.0. The optimization procedure could be expanded to determine the precise pepsin to antibody weight ratio by testing other ratios (1:10, 1:20, 1:30, 1:40, 1:50). Results presented here are for monoclonal mouse IgG2a antibody. Antibody isotype and animal species cause a high degree of variation in digestion conditions and must be accounted for in other digestion experimentation. For example, pepsin digestion of mouse IgG1 antibodies is very inefficient, and most often is performed with ficin instead [1]. It is important to determine optimal conditions for digestion either by literature search or optimization experiments similar to ones described here due to the high costs of monoclonal antibodies and the variability of conditions. In this study we describe optimal conditions for F(ab ) 2 production through pepsin digestion of IgG2a mouse monoclonal antibodies. Figure 3. (a) SDS-PAGE gel showing ph optimization. (b) Results from gel analysis showing relative band intensity versus digestion ph with whole IgG2a band (grey) and F(ab ) 2 band (black).
Figure 4. (a) SDS-PAGE gel showing pepsin-to-antibody weight ratio optimization. (b) Results from gel analysis showing relative band intensity versus pepsin-to-antibody weight ratio with whole IgG2a (grey) and F(ab ) 2 (black). Additionally, a method is presented for conducting similar optimizations for IgG from other species. Findings presented here will allow for more economical and efficient production of F(ab ) 2 for research techniques and immunotherapeutic applications. ACKNOWLEDGEMENTS This work was supported by NSF grant 0647918. The authors would like to acknowledge the entire Bryers lab group for assistance with the study. REFERENCES 1. Bryers JD, Medical biofilms. Biotechnol and Bioengineering 2008; 100: 1-18. 2. Lamoyi E. Preparation of F(ab')2 fragments from mouse IgG of various subclasses. Methods in Enzymology 1986; 121: 652-663. 3. Lodish H, Molecular Cell Biology. New York. W. H. Freeman and Company; 2008; 1060-1068. 4. Pierce Protein Research Products F(ab ) 2 Preparation Kit. Mar 2009. http://www.piercenet.com/products/browse. cfm?fldid=01010503 5. Rousseaux, J., Rousseaux-Prevost, R., Bazin, H. Optimal conditions for the preparation of proteolytic fragments from monoclonal IgG of different rat IgG subclasses. Methods in Enzymology. 1986, 121: 663-669.