How To Make An Antibody Fragmentation

Transcription

1 Antibody Simplify Engineering p plify Life Scien Sci e y Life Scie fy Life Sci

2 IgG Fragmentation FabRICATOR Antibody Fragmentation - Rapid, recise and Gentle Utilizing the unique specificity of a novel proteolytic enzyme, Genovis has developed a product to rapidly generate precise fragments. FabRICATOR cleaves IgG at a singular defined site below the hinge region to yield fragments in minutes. Why FabRICATOR? Unmatched yield and reaction to reaction consistency. Complete cleavage of IgG to fragments in minutes. ysiological reaction conditions preserves 100 % immunoreactivity of produced fragments. Homogeneity - only one cleavage site ensures that all fragments are identical. S C D K T H T C C A E L L G G S V FabRICATOR - Species cleaved Human IgG Mouse IgG1 Humanized IgG Mouse IgG2a* Chimeric IgG Mouse IgG2b Monkey IgG Mouse IgG3 Rabbit IgG Bovine IgG Sheep IgG orcine IgG Rat IgG Goat IgG * Special incubation conditons required MW (kda) Lane 1: Molecular weight marker Mark 12 (Invitrogen) Lane 2: IgG fraction Lane 3: 5 min digestion with FabRICATOR Lane 4: 15 min digestion with FabRICATOR Lane 5: 30 min digestion with FabRICATOR Lane 6: 30 min digestion with FabRICATOR (reduced sample) Lane 7: After removal of Fc fragments Lane 8: After removal of FabRICATOR Lane 9: Molecular weight marker Mark 12 SDS-AGE Enzyme Type ph Optimum Activators Inhibitors pi Advantages Disadvantages epsin Acid 1 (1-5) --- ph > 6, epoxides apain Thiol 6.5 (4-9.5) Cystein, sulfides, sulfite, cyanide, EDTA Ficin Thiol 6.5 (4-9.5) Cystein, sulfides, sulfite, cyanide, EDTA Heavy metals, carbonyls, NEM Heavy metals, carbonyls, NEM 11 Specific, easy to control digestion, no thiol required 1.5 Neutral ph digestion, broad species range, produces Fab and 1.5 Neutral ph digestion, broad species range, produces Fab and FabRICATOR Iodoacetate Fast, specific, neutral ph, yield, no over digestion Acid ph = low immunoreactivity, requires optimization Requires thiol concentration optimization, Mixture of Fab and Requires thiol concentration optimization, Mixture of Fab and Limited species range

3 FragIT - Spin Columns FragIT Spin column allows for easy and quick fragmentation of IgG into and Fc fragments. Fragmentation of large amounts of IgG is typically associated with low yield, time consuming optimization and operation at low ph with loss of immunoreactivity as a consequence. With FragIT spin columns and Fc fragments are generated at physiological ph with high purity and yield. The columns come prefilled with FabRICATOR enzyme immobilized on agarose beads for fragmentation of up to 100mg IgG. recise and Fc fragments are collected after column operation with no need to remove the enzyme. and Fc fragments can be separated with CaptureSelect, size exclusion or ion exchange chromatography. Spin columns are available in different sizes. Equillibrate Load & Incubate Spin & Collect SDS-AGE Lane 1: Molecular weight marker Lane 2: Human monoclonal IgG (Herceptin ) Lane 3: Human monoclonal IgG (Herceptin ) and Fc FragIT Kit - Some Things are Simple With FragIT Kit IgG is fragmented and and Fc fragments separated in a quick two-step process with very high yield. After fragmentation on FragIT spin column, and Fc fragments are separated on a Fc capturing spin column. The CaptureSelect column contains anti Fc IgG conjugated to agarose beads. Fc fragments are trapped on the beads while are passing through the column and collected. Fc fragments can be eluted from the agarose beads if desired. FragIT Kit contains one spin column for IgG fragmentation (FragIT spin column) and one Fc capturing spin column. The kit is available in three standard sizes; 0.5, 10 and 100 mg. Genovis provides other formats on request. If Fab fragments is a more desirable format the conversion into Fab fragments is performed in one quick reduction step, for further information see web site SDS-AGE Lane 1: Novex Sharp re-stained rotein Standards (Invitrogen) Lane 2: Avastin Lane 3: : After fragmentation using FragIT MicroSpin column Lane 4-6: Flow-through after removal of Fcfragment using CaptureSelect* Fc-specific resin. *CaptureSelect is a trademark of BAC BV, Netherlands.

4 IgG Deglycosylation IgGZERO - Antibody deglycosylation Deglycosylation of IgG molecules is an effective tool to minimize side effects in various immunoassays due to complement binding or cross-reactivity. The effects mediated by FcγRs are of great interest in the study of immunology. In vitro evaluation of such effects is complicated because cells isolated from subjects or individuals are typically pre-loaded with IgG bound to their FcγRs. Accordingly the receptors are blocked, preventing further analysis. IgGZERO Deglycosylate native IgG Human IgG Monkey IgG Mouse IgG Rabbit IgG Sheep IgG Rat IgG Goat IgG orcine IgG And others Why IgGZERO? kda 62 A B C D First known bacterial enzyme with a unique specificity for native IgG. Removal of IgGZERO is extremely easy by the included histidine tag in the amino terminal of the recombinant enzyme. Efficiently strip bound IgG from FcγRs without causing additional damage to native cells Stain Blot SDS-AGE and lectin blot analysis of IgG incubated with (+) or without (-) IgGZERO. Lanes A and C, IgG incubated with IgGZERO; lanes B and D, IgG incubated with BS alone. Add IgGZERO Wash rimary cells carrying IgG molecules in the Fcγ receptors deglycit Spin Column deglycit Spin column allows for easy and quick deglycosylation of IgG. deglycit Spin column comes prefilled with IgGZERO enzyme immobilized on agarose. deglycit Spin column is designed for deglycosylation of up to 100 mg IgG and is available in different sizes SDS-AGE Lane 1: Before deglycosylation of human monoclonal IgG (Herceptin ) Lane 2: Molecular weight marker Lane 3: After deglycosylation of human monoclonal IgG (Herceptin )

5 Mass Spec Kit When performing mass spectrometry of large proteins, such as IgG molecules, there is often a need to remove the glycans because of poor quality of the data otherwise obtained. Additionally, fragmentation of IgG can visualaize individual parts of IgG and allow for studies in more detail. The Mass Spec Kit contains both the FabRICATOR and IgGZERO enzyme for performing deglycosylation and fragmentation of 1 mg IgG in one hour. Mass spectrometry of samples can then be run directly without further purification. Effect of deglycosylating IgG Fc Without IgGZERO With FabRICATOR With IgGZERO With FabRICATOR Mass spectrometry of Fc-fragments from human polyclonal IgG purfied from plasma. (A) 1 mg of IgG was incubated with 1000 U of IdeS for 30 minutes and analyzed by mass spectrometry. (B) 1 mg of human IgG was incubated with 1000 Units of IdeS for 30 minutes followed by incubation with 1000 Units of EndoS for 45 minutes and finally analyzed by mass spectrometry. Utilization of RC18 LC-ESI-MS generated a well-defined protein envelope with a deconvoluted mass of Da corresponding to the deglycosylated IgG1 heavy chain cleaved at position Gly236. Neuα2 6Galβ1 4GlcNAcβ1 2Manα1 GlcNAcβ1 Neuα2 6Galβ1 4GlcNAcβ1 2Manα1 6 4Manβ1 3 Fucα1 4GlcNAcβ1 6 4GlcNAc Asn-297 IgGZero cleaves of the side chain glucans leaving the first core N-acetyl-D-glucosamine, or GlcNAc, attached to Asn-297. It is further possible to analyse the degree of fucosylation, since IgGZero leaves the fucose intact in case the GlcNAc is fucosylated. Homogeneity of fragments epsin vs FabRICATOR epsin FabRICATOR Mass spectrometry (LS/MS) of -fragments from humanized monoclonal IgG (Hercptinγ). (A) 1 mg of IgG was digested using epsin (Sigma) for 2 hours ph 3.5. fragments was isolated by size exclusion chromatograpy before analyzed by mass spectrometry. (B) mg of IgG was digested using FabRICATOR for 30 minutes and fragments was isolated using size exclusion chromatograpy before analyzed by mass spectrometry.

6 Antibody Engineering Description Size Item # FabRICATOR, Bulk quantity Bulk A0-FR1-999 FabRICATOR, Cleaves 25 mg IgG 25000u A0-FR1-250 FabRICATOR, Cleaves 5 mg IgG 5000u A0-FR1-050 FabRICATOR, Cleaves 2 mg IgG 2000u A0-FR1-020 FragIT MicroSpin, Cleaves up to 0,5 mg IgG 2 columns A0-FR6-010 FragIT MicroSpin, Cleaves 5 x 0,5mg 5 columns A0-FR6-025 FragIT MicroSpin, Cleaves 10 x 0,5mg 10 columns A0-FR6-050 FragIT MidiSpin, Cleaves 1-10mg 1 column A0-FR6-100 FragIT MaxiSpin, Cleaves mg 1 column A0-FR DOCKSIDE.SE maj FragIT Kit, Cleave & purify up to 0.5 mg IgG 1+1 micro spin column A2-FR2-005 FragIT Kit, Cleave & purify up to 5 x 0.5 mg IgG 5+5 micro spin columns A2-FR2-025 FragIT Kit, Cleave & purify up to 10 mg IgG 1+1 midi spin columns A2-FR2-100 FragIT Kit, Cleave & purify up to 100 mg IgG 1+1 maxi spin column A2-FR IgGZERO, Bulk quantity Bulk A0-IZ1-999 IgGZERO, Cleaves 25 mg IgG 25000u A0-IZ1-250 IgGZERO, Cleaves 5 mg IgG 5000u A0-IZ1-050 IgGZERO, Cleaves 1 mg IgG 1000u A0-IZ1-010 deglycit MicroSpin, Cleaves up to 0,5 mg IgG 2 columns A0-IZ6-010 deglycit MicroSpin, Deglycosylates 5 x 0,5mg 5 columns A0-IZ6-025 deglycit MicroSpin, Deglycosylates 10 x 0,5mg 10 columns A0-IZ6-050 deglycit MidiSpin, Deglycosylates 1-10mg 1 column A0-IZ6-100 deglycit MaxiSpin, Deglycosylates mg 1 column A0-IZ Mass Spec Kit 1000u A0-IZ2-010 Contact: l phone: Culver Road, Suite 307 Rochester, NY im