Biopharmaceutical Characterization Analysis Tool: Enzymes

Background

Therapeutic antibodies serving as biopharmaceuticals possess intricate structures and exhibit significant heterogeneity, necessitating rigorous oversight and regulation throughout the manufacturing process. Heterogeneity in antibody molecular size is a crucial quality control metric for optimizing the production process, controlling the manufacturing steps, and conducting release analysis. To uphold the safety and effectiveness of monoclonal antibodies and other biomolecule drugs, a thorough and profound characterization of these medications is imperative.

As the number of approved antibody drugs, including mAbs, Fc fusion proteins, Fabs, ADCs, bispecific antibodies, and bispecific T cell engagers (BiTEs), grows exponentially each year, gaining insights into individual post-translational modifications (PTMs) can aid in better analyzing and predicting the in vivo efficacy, pharmacokinetics, and mechanisms of action of therapeutic antibodies.

Characterization of Therapeutic Antibodies and Fusion Proteins

The characterization of therapeutic antibodies and fusion proteins is typically conducted at three distinct levels: top-down for complete molecular weight analysis, bottom-up for peptide-level analysis, and at the subunit level. The commonly utilized tool enzymes for the physicochemical analysis of therapeutic proteins include PNGase F enzyme, IdeS enzyme, Papain, GinsKHAN, GlySERIAS enzyme, trypsin for peptide mapping, GluC, LysC, chymotrypsin, and more.

Seebio offers a comprehensive range of antibody/protein physicochemical analysis tool enzymes that aid in the characterization and analysis of monoclonal antibodies, fusion protein drugs, biosimilars, and other biopharmaceuticals, including their associated post-translational modifications. These enzymes support the development and quality control of biopharmaceuticals.

Protein & Peptide Mapping Analysis

Protease	Restriction Sites	Characteristics
Recombinant Trypsin (mass spectrometry grade）	Carboxy terminus in lysine and arginine residues	Trypsin Type proportion >80% Specific activity ≥6200 USP U/mg (equivalent to 18600 BAEE U/mg) Exceptional stability, withstanding up to 10 freeze-thaw cycles. Compared to imported brands, it exhibits a lower peptide miss rate and a higher number of identified proteins. Consistent batch-to-batch performance, with relevant regulatory support documents available.
Recombinant Lysyl Endonuclease (Lys-C)	Amide, ester and peptide bonds on the carboxyl side of lysine	Used alone or in combination with trypsin to identify more proteins and phosphorylation sites. Maintains high enzyme activity in the presence of 4M urea or 0.1% SDS.
Glu-C protease	Glutamic acid or aspartic acid residue carboxyl terminal peptide bond	For proteins with high lysine and arginine content, combining with trypsin significantly increases the number of identified proteins. Suitable for isolation and enrichment of phosphorylated peptides.
Recombinant Humana-α-chymotrypsin	Carboxyl-terminal peptide bonds of tyrosine, tryptophan, and phenylalanine	Particularly suitable for membrane protein analysis.

Glycosylation Analysis Enzymes

Glycosidase	Name	Restriction sites	Characteristics
Sialic Acid Modification	Sialidase (a2-3,6,8)	Sialic acid linked via a2-3, a2-6, a2-8 bonds on glycoproteins and oligosaccharides	Can act on both N-glycolylneuraminic acid and N-acetylneuraminic acid.
	Sialidase (a2-3,6,8,9)	Sialic acid linked via a2-3, a2-6, a2-8 and a2-9 bonds on glycoproteins and oligosaccharides	Can act on both N-glycolylneuraminic acid and N-acetylneuraminic acid； Tolerates 0.5%-1.0% detergent.
N-glycosylation Modification	PNGase F	Glycosidic bond between asparagine and the innermost GICNAc	Suitable for complete N-glycosyl removal of antibodies and glycoproteins. Can cleave glycoproteins under denaturing and non-denaturing conditions.
	Recombinant Endo H	High mannose in N-glycoproteins and the chitobiose core structure of some hybrid oligosaccharides	Suitable for complete removal of N-linked high mannose from antibodies or other glycoproteins. Storage solution does not contain glycerol.
O-glycosylation Modification	O-glycoproteinase	Serine or threonine residues of mucin-type O-linked sugars (sialylated or not)	Suitable for O-glycosylation analysis; no sialidase pretreatment required.

Antibody Cleavage Enzyme

Antibody Cleavage Enzyme

IdeS Protease and IdeZ Protease are recombinant proteases originating from Streptococcus pyogenes and Streptococcus equi, respectively. These proteases exhibit exceptionally high substrate specificity, recognizing specific species such as humans and a distinct type of IgG. They catalyze enzymatic digestion at a precise site below the antibody hinge region, resulting in the hydrolysis of IgG into a complete F(ab')2 fragment and an Fc fragment. The IdeS/Z enzyme serves as an essential tool enzyme in the preparation and structural characterization analysis of antibody drugs. It is invaluable for characterizing therapeutic antibodies, monoclonal antibodies, antibody-drug conjugates, Fc fusion proteins, and antibody-drug complexes.

Name	Restriction sites	Characteristics
IdeS protease	Specific recognition of IgG and at specific sites in the lower hinge region of IgG	Can hydrolyze IgG into complete F(ab')2 fragment and Fc fragment.
IdeZ protease	Specific recognition of IgG and at specific sites in the lower hinge region of IgG	Can act on both N-glycolylneuraminic acid and N-acetylneuraminic acid； Can tolerate 0.5%-1.0% detergent.

Tag Excision Enzyme

Name	Restriction sites	Characterisitcs
Recombinant TEV protease (rTEV Protease）	Strictly identify the seven amino acid sequence EXXYXQ↓(G/S) cleavage site between glutamine and glycine/serine.	A versatile tool enzyme for excising tag sequences from fusion proteins, offering high activity and specificity compared to proteases like EK and SUMO.

Seebio Protein Enzymes

Protein Sequencing Peptide Analysis
Item number	Product	Packaging
DCE0060U	Recombinant trypsin (mass spectrometry grade, liquid）	25 ug 100 ug
DCE0060T	Recombinant trypsin (mass spectrometry grade, lyophilized）	25 ug 100 ug
EXK0369B	Recombinant lysyl endonuclease	20 ug
AXJ4266C	Recombinant Glu-C protease, lyophilized	25 ug 100 ug
AXJ4266B	Recombinant Glu-C protease	20 ug 100 ug
DCL0640B	Recombinant human a-Chymotrypsin (lyophilized）	20 ug 50 ug 100 ug
Glycosylase
>ECE0441E	PNGase F recombinant glycosidase	20KU 40KU
DCE0447C	Recombinant Endo H	3KU 15KU
DCE0448B	O-glycoproteinase	20u 100u
DCE0457F	sialidase(a2-3,6,8)	500u 2KU 10KU
DCE0457E	sialidase(a2-3,6,8,9）	2KU 10KU
Antibody Cleavage Enzyme
ECE0711A	IdeS protease	700u
ECE0712A	IdeZ protease	1KU 2KU
Tag Excision Enzyme
EBY3644A	Recombinant tobacco virus protease (rTEV Protease）	1KU 5KU 10KU