APR Sept/Oct 2023 - 53

« MICROBIOLOGY
demonstrated an important component in the clotting cascade that
is responsible for the LAL test reaction (and for the immune response
of the horseshoe crab).7
This was called factor C proenzyme and it was
experimentally demonstrated that factor C was the only biosensor
that detected endotoxins, thus establishing the cascade reaction.
With the clotting reaction, activated factor C activates factor B, which
in turn activates a pro-clotting enzyme to form a corresponding
clotting enzyme. The clotting enzyme hydrolyzes a specific site of a
coagulogen molecule and a coagulin gel is formed, and thus the lysate
is coagulated. For the LAL/TAL test, endotoxin is measured through
measuring the lysate coagulation reaction. In an unmodified form
of the lysate, another factor (factor G) was shown to also generate a
cascade response in the presence of beta glucans.8
The modified recombinant assay is based on a simple single reaction
that involves the cleaving of a fluorogenic substrate by factor C. The
simplified assay does not require the remaining proteins that make up
the LAL cascade, such as factor B, factor G and proclotting enzyme.
Hence, alongside the simplicity is greater assay specificity. Other
endotoxin test assays use recombinant protein technology to replicate
the complete pathways from the point of endotoxin detection to
create reagents that are closer to horseshoe crab lysate.
Recombinant Reagent
Manufacturing Process
In 1997 a bacterial endotoxin test based on recombinant factor C (rFC)
was demonstrated experimentally and reported.9
Following this, in
2004, a working reagent with commercial potential was developed.
This was rFC, as an animal-free alternative to the endotoxin sensor
protein inside of Limulus amoebocyte lysate (LAL). As well as having
the same functionality as horseshoe crab lysate, recombinant factor
C does not recognize the factor G pathway; hence the product can
detect endotoxin more selectively.10
Since these initial developments
numerous studies have compared LAL and rFC in terms of bacterial
endotoxin detection.11-14
Generally, the outcome of such studies has
been favorable, although method validation for each type of sample
(rather than verification) is advised.
With the recombinant reagent (either rFC or a wider pathway), a
recombinant protein is an artificially produced (and often purified)
protein, generated from the appropriate gene from one of the four
known living species of horseshoe crab. The recombinant process
occurs when recombinant DNA encoding a protein is introduced into
a host organism to express foreign proteins. This process requires the
use of specialized expression vectors together with restructuring
by foreign coding sequences.14,15
Since the resultant reagent is not
derived from horseshoe crab amoebocytes it cannot be called LAL
or TAL.
The first recombinant factor C reagents were produced based on
biosynthesis using yeasts.16
In the early stages the manufacture
proved variable, in trying to produce rFC as an approximate 132
kDa molecule in the form of a zymogen/proenzyme (a biologically
inactive substance which is metabolized into an enzyme). The first
larger scale rFC was based on the cloned cDNA sequence of factor C
»
from the mangrove horseshoe crab Carcinoscorpius rotundicauda and
expressed initially in insect cells with other methods subsequently
developed, demonstrating the appropriate productivity, bioactivity,
and physicochemical characteristics.17
High-throughput screening,
design-of-experiments, and other analytical advancements paved the
way for recombinant factor C (and for replicating other parts of the
clotting pathway) production to scale.
Given that the protein secretory pathway is an important pathway for
eukaryotic cells, therefore, based on protein synthesis capabilities,
eukaryotic cell lines are used widely for the process of producing
recombinant proteins. Mammalian cell systems for recombinant
protein production offer diverse advantages. These include good
protein folding, absence of many potentially immunogenic posttranslational
modification products, good secretion capacity, and a
medium to high product yield. The important considerations required
during cell-line development include selection of an expression host,
vectors, and transfection, and cell-line selection.
Recombinant protein production begins with expression vector
engineering and transfection into a host system. This step is followed
by the steps of:18
* Cell selection
* Medium selection (defining the essential nutrients required
for optimal cell growth and target protein productivity is very
important)
* Cloning
* Screening
* Evaluation
The objective of manufacturing is the standardized production of
the same rFC protein through the use of a bioreactor. This requires
achieving a certain level of quality and scalability. Important criteria
for an optimal cell line include:19
* High cell density in suspension culture.
* Key cultivation parameters, such as media composition,
pH, agitation, aeration, temperature, cell density,
the concentration of inducers, induction time, and
feeding strategies.
* Suitable bioreactor methods, with batch, fed-batch (where
nutrients are fed during cultivation), and continuous or
perfusion culture (where the culture is fed through the medium
in order to streamline both waste removal and nutrient supply),
being available for bulk production of recombinant proteins.
»
The type of bioreactor is also very important. The types
of models available include: stirred tank bioreactors,
airlift bioreactors, membrane bioreactors, bubble
column bioreactors, hollow fiber bioreactors, and fixed
bed and fluidized bed bioreactors.
Operational parameters for bioreactors include:
Agitation power settings, maintaining the oxygen
transfer coefficient, consistency of continual mixing
times, agitation impeller speed, control of the heat
www.americanpharmaceuticalreview.com |
| 53
»
http://www.americanpharmaceuticalreview.com
APR Sept/Oct 2023

Table of Contents for the Digital Edition of APR Sept/Oct 2023
