CELL CULTURE

rTrypsin

RethinkTomorrow

Graphical representation of rTrypsin molecule

Trypsin Trypsin (EC 3.4.21.4) is an endopeptidase that preferentially hydrolyses ester bonds whose carboxyl groups are

contributed by Lysine (Lys) or Arginine (Arg) except when either is followed by proline (PRO). It is used for numerous

biotechnological processes such as tissue dissociation, cell harvesting and subculturing cells. Although native trypsin from

bovine or porcine pancreas has been available for many years, more stringent regulations are now engaged to ensure

safer biologicals and biopharmaceuticals. Novozymes offers rTrypsin, a recombinant product manufactured by microbial

fermentation. rTrypsin is animal component-free and manufactured to ISO 9001:2008 standards.

Advantages of rTrypsin• Produced by a recombinant manufacturing process, rTrypsin is not contaminated with animal enzymes such as

chymotrypsin or carboxypeptidases.

• Animal origin-free (AOF) manufacturing process eliminates the risk of viral and animal-related contaminants in

the final product

• Allows simple replacement of bovine or porcine trypsin

• Stability is not affected by Ca2+

• Consistent supply

Applications for rTrypsin• Cell dissociation and propagation of multiple cell lines including stem cells

• Vaccine production and processing

• Dissociation of dissected cells prior to cell fixing and sorting

• Cleavage of proinsulin to yield intact insulin

StabilityrTrypsin is available as a granulated product.

Temperature stabilityThe exceptional stability of rTrypsin permits easy and convenient storage and routine handling of the enzyme during

cell culture procedures.

Stability of rTrypsin is not affected by

free Ca2+ ions in the buffer

Excess free Ca2+ (1mM CaCl2)Without excess free Ca2+ (+1mM EDTA)

Excess free Ca2+ (1mM CaCl2)Without excess free Ca2+ (+1mM EDTA)

Stability of porcine trypsin is dependent on

free Ca2+ ions in the buffer

Rel

ativ

e ac

tivi

ty

Rel

ativ

e ac

tivi

ty

SpecificityrTrypsin cleaves peptide bonds at the carboxyl side of

lysine and arginine as native trypsin. This permits simple

substitution of rTrypsin for porcine or bovine trypsin.

rTrypsin for dissociating cellsrTrypsin may be used to re-suspend cells from monolayers grown adhering to the interior surfaces of culture vessels.

rTrypsin performs well on stem cell lines such as iPH and hES as well as CHO, Vero and MDCK cells lines. Cells are most

commonly removed from the culture substrate by treatment with rTrypsin/EDTA/PBS buffer adjusted to pH 7. Depending

on the cell line, the suggested rTrypsin concentration should be adjusted to 0.2 mg/ml (corresponding to 200 U/ml

rTrypsin with an activity of 1000 USP/mg powder) and sterile filtered.

Available from Novozymes

USP = Trypsin activity unit using USP Crystallised Trypsin Reference Standard. The activity is determined relative to a protease A standard.

pH stability pH activity

rTrypsin is active at physiological pH 7.4, at 37°CrTrypsin is stable for 2hrs. at 37°C, pH range 5-9

rTrypsin Specificity at pH 9.0 on Suc-AAPX-pNA

Substrates: Suc-AAPA-pNA, Suc-AAPR-pNA,Suc-AAPD-pNA, Suc-AAPI-pNA, Suc-AAPM-pNA,Suc-AAPV-pNA, Suc-AAPL-pNA, Suc-AAPE-pNA,Suc-AAPK-pNA, Suc-AAPF-pNA.

Assay buffer: 100mM succinic acid, 100mM HEPES,100mM CHES, 100mM CABS, 1mM CaCl2,150mM KCl, 0.01% Triton X-100, pH 9.0.

Assay: 20μl rTrypsin/porcine trypsin(diluted in 0.01% Triton X-100) was mixed with100μl assay buffer in a microtiterplate well.The assay was started by adding 100μl pNA substrate(50mg pNA-substrate dissolved in 1.0ml DMSO andfurther diluted 45x with 0.01% Triton X-100).

The increase in OD405 was monitored as a measureof rTrypsin/porcine trypsin activity.

SA181 (hESC) after five passages using rTrypsin. Fixated cells were stained with an antibody detecting Oct4, a typical marker used to detect undifferentiated cells.

Product Name Product No. Unit size

rTrypsin 6395020 100g or 1 kg

For Research Use or Manufacturing of Cell, Gene, or Tissue-Based Products. CAUTION: Not intended for direct administration into humans or animals.

Product Name EC No. Substrate specificity Format Optimal Conditions Unit Activity Application

rTrypsin 3.4.21.4 Serine protease granulate pH 7.8-8.0 800 USP/mgHydrolysis of amide and ester bonds of lysine and arginine at carboxyl terminal

Rel

ativ

e ac

tivi

ty

Rel

ativ

e ac

tivi

ty

Rel

ativ

e ac

tivi

ty

Novozymes Biopharma US Inc.(reg no. 01142208)77 Perry Chapel Church RoadFranklinton, NC 27525, USATel. +1 919 494 3000

Novozymes Biopharma DK A/SKrogshoejvej 362880 Bagsvaerd,DenmarkTel: +45 4446 2896

Novozymes is the world leader in bioinnovation.

Together with customers across a broad array of industries

we create tomorrow’s industrial biosolutions, improving our

customers’ business and the use of our planet’s resources.

Read more at www.novozymes.com.

The products and services described in this document are the responsibility of Novozymes Biopharma DK A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark (company registration no. 29603537) -

a wholly owned subsidiary of Novozymes A/S. The information in this document is based on data we believe to be reliable. They are offered in good faith, but without warranty, as conditions and

methods of use of the products are beyond our control. Furthermore, laws, regulations, and/or third-party rights may prevent the recipient from using the information herein in a given manner.

Thus, the information contained herein is provided “AS IS” and Novozymes makes no representation or warranty whatsoever with regard to said information, hereunder the accuracy, fitness for a

particular purpose, non-infringement of intellectual property rights, or regulatory/legal compliance, unless otherwise agreed in writing.

© Novozymes A/S · No. 2014-12716-01