Literature summary for 3.4.22.15 extracted from

Luo, H.; Tie, L.; Cao, M.; Hunter, A.K.; Pabst, T.M.; Du, J.; Field, R.; Li, Y.; Wang, W.K.
Cathepsin L causes proteolytic cleavage of chinese-hamster-ovary cell expressed proteins during processing and storage identification, characterization, and mitigation (2019), Biotechnol. Prog., 35, e2732 .

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Inhibitors

Inhibitors	Comment	Organism
bestatin	weak inhibition	Cricetulus griseus
E64	strong inhibition	Cricetulus griseus
leupeptin	strong inhibition	Cricetulus griseus
additional information	no inhibition by EDTA, AEBSF, approtinin, and pepstatin A	Cricetulus griseus

Organism

Organism	UniProt	Comment	Textmining
Cricetulus griseus	G3INC5	-	-

Purification (Commentary)

Purification (Comment)	Organism
cathepsin L exists in Chinese hamster ovary cell culture fluids obtained from cell lines expressing different products and cleaves a variety of recombinant proteins including monoclonal antibodies, antibody fragments, bispecific antibodies, and fusion proteins. Product fragmentation is caused by copurification of the cysteine protease, Cathepsin L. Therefore, characterization its chromatographic behavior is essential to ensure robust manufacturing and sufficient shelf life. The chromatographic behaviors of Cathepsin L using a variety of techniques including affinity, cation exchange, anion exchange, and mixed mode chromatography are systematically evaluated. Copurification of cathepsin L on nonaffinity modalities is principally because of similar retention on the stationary phase and not through interactions with product. Cathespin L exhibits a broad elution profile in cation exchange chromatography (CEX) likely because of its different forms. Affinity purification is free of fragmentation issue, making affinity capture the best mitigation of cathepsin L. When affinity purification is not feasible, a high pH wash on CEX can effectively remove cathepsin L but results in significant product loss, while anion exchange chromatography operates in flow-through mode does not efficiently remove cathepsin L. Mixed mode chromatography, using adsorption resin in this example, provides robust clearance over wide process parameter range (pH 7.7, 50 mM NaCl), making it an ideal technique to clear cathepsin L, method developent and evaluation, overview. Fragmentation does not occur at pH 7.0 at both 25°C and 37°C, suggesting that the protease is nearly inactive under neutral pH conditions. At pH 5.0 and pH 6.0, while the fragmentation is barely detectable at 25°C, it is pronounced at 37°C. The results suggest that the presence of the proteolytic activity under mildly acidic conditions (commonly used for formulation) still poses a significant threat to long-term product stability	Cricetulus griseus

Purification (Comment)

Organism

cathepsin L exists in Chinese hamster ovary cell culture fluids obtained from cell lines expressing different products and cleaves a variety of recombinant proteins including monoclonal antibodies, antibody fragments, bispecific antibodies, and fusion proteins. Product fragmentation is caused by copurification of the cysteine protease, Cathepsin L. Therefore, characterization its chromatographic behavior is essential to ensure robust manufacturing and sufficient shelf life. The chromatographic behaviors of Cathepsin L using a variety of techniques including affinity, cation exchange, anion exchange, and mixed mode chromatography are systematically evaluated. Copurification of cathepsin L on nonaffinity modalities is principally because of similar retention on the stationary phase and not through interactions with product. Cathespin L exhibits a broad elution profile in cation exchange chromatography (CEX) likely because of its different forms. Affinity purification is free of fragmentation issue, making affinity capture the best mitigation of cathepsin L. When affinity purification is not feasible, a high pH wash on CEX can effectively remove cathepsin L but results in significant product loss, while anion exchange chromatography operates in flow-through mode does not efficiently remove cathepsin L. Mixed mode chromatography, using adsorption resin in this example, provides robust clearance over wide process parameter range (pH 7.7, 50 mM NaCl), making it an ideal technique to clear cathepsin L, method developent and evaluation, overview. Fragmentation does not occur at pH 7.0 at both 25°C and 37°C, suggesting that the protease is nearly inactive under neutral pH conditions. At pH 5.0 and pH 6.0, while the fragmentation is barely detectable at 25°C, it is pronounced at 37°C. The results suggest that the presence of the proteolytic activity under mildly acidic conditions (commonly used for formulation) still poses a significant threat to long-term product stability

Cricetulus griseus

Source Tissue

Source Tissue	Comment	Organism	Textmining
CHO cell	-	Cricetulus griseus	-
ovary cell line	-	Cricetulus griseus	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
FabA antibody + H2O	model protein, determination of fragments compared to the recombinant murine enzyme	Cricetulus griseus	?	-	?
additional information	owing to its low specificity, cathepsin L is able to cleave a diversity of proteins, it cleaves a variety of recombinant proteins including monoclonal antibodies, antibody fragments, bispecific antibodies, and fusion proteins. Cathepsin L is a low specificity protease. CHO cathepsin L seems to have preferential cleavage sites on mAbs of different subclass as similar fragment profiles exist for three tested mAbs. IgG1 does not interact with cathepsin L	Cricetulus griseus	?	-	?

Subunits

Subunits	Comment	Organism
More	three forms of molecular weight of 34, 32, and 28 kD, respectively, SDS-PAGE	Cricetulus griseus

Synonyms

Synonyms	Comment	Organism
cathepsin L	-	Cricetulus griseus
cathepsin L1	-	Cricetulus griseus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Cricetulus griseus

Temperature Range [°C]

Temperature Minimum [°C]	Temperature Maximum [°C]	Comment	Organism
additional information	-	protein fragmentation by the enzyme does not occur at pH 7.0 at both 25°C and 37°C, suggesting that the protease is nearly inactive under neutral pH conditions. At pH 5.0 and 6.0, while the fragmentation is barely detectable at 25°C, it is pronounced at 37°C	Cricetulus griseus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
3.4	-	assay at	Cricetulus griseus

pH Range

pH Minimum	pH Maximum	Comment	Organism
3	6.5	activity range. Protein fragmentation by the enzyme does not occur at pH 7.0 at both 25°C and 37°C, suggesting that the protease is nearly inactive under neutral pH conditions. At pH 5.0 and 6.0, while the fragmentation is barely detectable at 25°C, it is pronounced at 37°C	Cricetulus griseus