PyroSmart NextGen™

Meet your sustainability objectives with Recombinant Cascade LAL Reagent

After Focusing on supporting fisheries worldwide and ensuring the genetic diversity of the horseshoe crab (read more), our partner Associates of Cape Cod, Inc. (ACC), has launched PyroSmart NextGen™.

This Recombinant LAL Cascade Reagent works exactly the same, but performs over all better then classic LAL reagent. Furthermore, it is animal free.

The origin

Throughout the past decade, in an effort to continually improve BET incorporating current technological advances, the PyroSmart® reagent (based on genetic sequence of the Japanese horseshoe crab Tachypleus tridentatus (TAL)) was developed and characterized as the first recombinant Cascade Reagent (rCR).

Later on, PyroSmart NextGen™ was introduced which builds on the success and analytical performance of its predecessor but whose recombinant proteins are expressed based on the genetic sequence of Limulus polyphemus. This is due to the fact that LAL reagents, not TAL, are the primary reagents utilized by the vast majority of global end users and there are no FDA licensed TAL reagents for BET.

Utilizing the same LAL cascade as traditional LAL reagents, while eliminating the potential for 1,3-β-D-glucans cross reactivity, PyroSmart NextGen™ delivers all of the quality and consistency of results you have come to expect from ACC LAL reagents.

PyroSmart NextGen™ can be used for a wide variety of endotoxin tests, ranging from standard water testing to samples requiring high sensitivity, such as intrathecal products and those requiring high dilutions to overcome interference.

Product Sensitivity

The sensitivity for recombinant chromogenic assays is determined by the lowest standard concentration on the standard curve used for the assay. The maximum sensitivity of PyroSmart NextGen™ is 0.005 EU/mL when run in an incubating microplate reader (or 0.001 EU/mL when run in Pyros Kinetix® Flex tube reader).

Literature

2019 Muroi et al. Application of a Recombinant Three-Factor Chromogenic Reagent PyrosSmart for BET Filed in the Pharmacopeais.
2022 Stevens et al. Advanced Recombinant Cascade Reagent PyroSmart NextGen® for Bacterial Endotoxins Test as Described in the Pharmacopeias.
2022 Kelley et al. Evaluation of Recombinant Cascade Reagent PyroSmart NextGen® and Limulus Amebocyte Lysate Equivalency in a Plate and Tube Reader for Bacterial Endotoxins Testing.

Traditional vs Next generation

	Conventional LAL Reagent	PyroSmart NextGen™ (rCR)	Competitor (rFC) Reagent
Year Technology Introduced	1977	2021	2005
Kinetic Assay	Kinetic	Kinetic	Endpoint only
Assay Setup	Single step reconstitution	Single step reconstitution	Requires three reagents in a 1:4:5 ratio and a 10 min pre-incubation step
Same Standard Plate Reader	Incubating plate or tube reader at 405nm	Incubating plate or tube reader at 405nm	Fluorescent reader required
Derived From: Limulus Amebocyte Lysate (LAL)	LAL	rCR is recombinant LAL	Based on Carcinoscorpius or Tachypleus Amebocyte Lysate (CAL/TAL)
Multi-step Cascade Pathway	Yes	Yes	No
Endotoxin Specific	No	Endotoxin Specific	Endotoxin Specific
Sustainable Reagent (animal free)	No	Horseshoe Crab Blood Free	Horseshoe Crab Blood Free

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Frequently Asked Questions

1. Will PSNG perform the same as naturally sourced reagents?

Yes, PSNG is a kinetic chromogenic reagent. It contains recombinant Factor C, Factor B and pro-clotting enzyme, thus it continues to rely on the cascade mechanism in order to amplify the signal. It is co-lyophilized with the same chromogenic substrate as other chromogenic reagents. Therefore it is homogeneous with Pyrochrome® and Chromo-LAL. This presents a huge advantage to the end user: they keep the method, only switch out the reagent.

2. What’s the difference between Recombinant Factor C (rFC) and Recombinant Cascade Reagent (rCR)?

Recombinant Factor C reagent contains only recombinant Factor C. This enzyme acts as a biosensor for endotoxin (specifically binds to it). But since the cascade amplifying the signal is missing (no Factor B and pre-clotting enzyme), Factor C reagents are used in combination with fluorescence read out in order to achieve sensitivity levels comparable to the cascade reaction. This means that a fluorescent plate reader has to be used (along with a specific microplate). So this means different method, different readers, different SOPs for rFC reagents. Recombinant cascade reagent (e.g. recombinant TAL (= PyroSmart) or recombinant LAL (= PyroSmart NextGen™) are homogenous to current LAL reagents (Pyrochrome® or Chromo-LAL) in that they contain the full cascade (3-enzyme cascade).

3. Does PSNG use any raw material from the horseshoe crab?

No. PSNG is completely animal free.

4. Will new instrumentation be required to use PSNG?

No. Due to the cascade based feature, you can utilize the same instrument and preparation procedures.

5. How are recombinant reagents produced?

It is a biopharmaceutical production based on the use of expression system as cell line clones, that contain (thanks to genetic engeering) DNA coding Factor C, Factor B and pro-clotting enzymes from Limulus Polyphemus. The cell lines express the final glycoproteins Factor C, Factor B and pro-clotting enzyme which are then collected, purified and formulated into PyroSmart NextGen™.