Articles in this Issue
Kevin L. Williams
Bacterial Endotoxin Test (BET) users seek ways to overcome low
endotoxin recovery (LER) from direct spikes into undiluted biologics.
These studies have come about from Chen’s initial observation1 that
Control Standard Endotoxin (CSE) spikes, when placed into undiluted
biologics, buff ers, and other constituents often cannot be recovered.
By changing the endotoxin spike requirement from diluted product to
undiluted product, users will unsurprisingly encounter test interference;
what is unexpected from Chen’s LER fi nding is that recovery sometimes
cannot be improved by dilution. This is indicative of a still undefi ned
binding phenomenon.
Filippos Kesisoglou, Ph.D.
Dissolution experiments have been used for years as a quality control
tool for pharmaceutical products. The accompanying dissolution
specifications are intended to ensure consistency between formulation
batches during development and manufacturing. More recently,
increased attention has been placed on biorelevant dissolution.
Biorelevant dissolution, defined as dissolution in media designed
to mimic the composition of the gastrointestinal fluids [1], allows
for study of formulation behavior in an environment that could be
considered closer to the in vivo conditions compared to compendial
media, especially for BCS II/IV drugs. As such, biorelevant dissolution
could be seen as more useful in understanding the clinical implications
of any formulation changes.
Jeanne Moldenhauer
Recently, a number of different documents have been making
statements similar to “incubation conditions and media used must be
justified”. Many companies were able to cite USP as a rationale
for using two different incubation conditions including 20°C-25°C and
30°C-35°C. However, the most recent revision to USP states: “Time and
incubation temperatures are set once the appropriate media have
been selected. Typically, for general microbiological growth media
such as SCDM, incubation temperatures in the ranges of approximately
20°C–35°C have been used with an incubation time of not less than
72 hours.” [1]. With a 15°C range in temperature, it is not likely that
a single study will provide adequate data to justify the incubation
conditions use. As such, companies need to generate data to support
the incubation conditions utilized.
Stuart Farquharson, Ph.D.
Raman spectroscopy is becoming one of the most popular analytical measurement tools for
pharmaceutical applications ranging from verification of raw materials to process monitoring of
drug production to quality control of products. Similar to an infrared spectrum, a Raman spectrum
consists of a wavelength distribution of peaks corresponding to molecular vibrations specific to the
sample being analyzed (see Figure 1B). Chemicals, such as drugs, can be identified by the frequency
and quantified by the intensity of the peaks. In practice, a laser is focused into the sample, the
inelastic scattered radiation (Raman) is optically collected and directed into a spectrometer, which
provides wavelength separation, and a detector converts photon energy to electrical signal intensity.
An attractive advantage to this technique is that samples do not have to be extracted or prepared,
and the laser can simply be aimed at a sample to perform chemical measurements, which can often
be accomplished in a minute or less.
Laura Heikaus, Dr. Hartmut Schlüter
Displacement chromatography (DC) can be advantageous in
comparison to gradient chromatography. Parameters favoring DC
allow significantly larger sample loads since the binding capacity
can be exploited more efficiently
Michael Hodgkinson
The adoption of single-use technologies in drug product
manufacturing has introduced many benefits for the pharmaceutical/
biopharmaceutical industry. It has also introduced the need for an
entirely new way of thinking for a group of developing subject matter
experts to deal with a fresh set of challenges. Many of the advantages
of single-use technology have been well-characterized and discussed
in literature, including the reduction of cross-contamination risks
and cleaning validation program related resources, as well as faster
changeover times on the production floor, lower capital investment
costs on facility start-ups and less costs associated with routine
cleaning and sterilization of product contacting parts.
Actually, we use all four methods: gel-clot, kinetic turbidimetric,
kinetic chromogenic – and the PTS™. These methods are used
to test and release incoming raw materials and to evaluate intermediate
formulation buff ers that are subsequently used to produce licensed LAL
products. Our accessory products also benefi t from the use of these
methods prior to release. Finally, we leverage all of these techniques
in developing and validating LAL test methods for customer-specifi c
products.
Associates of Cape Cod, Inc.’s (ACC) fourth generation tube reader, the
Pyros® Kinetix Flex, offers the most sensitive bacterial endotoxin test
(BET) available for both turbidimetric and chromogenic kinetic methods.
In addition to flexibility of test method, ACC offers a choice of 32, 64 and
96 well readers. All of these readers provide the flexibility to add tubes
at any time. Unlike a microplate reader, additional samples can be added
after a test has been started.
The development of the endpoint chromogenic technology was a
critical step in the development of the kinetic chromogenic assays
that are in regular use today around the world. The discovery was
based on determining the sequence of amino acids at the cleavage
site of coagulin, the last step in the enzymatic cascade leading to gel
clot formulation in the classical gel-clot LAL assay. Once this sequence
was known, a peptide could be sequenced that could be attached to a
chromophore. When the chromophore was cleaved from the peptide,
a yellow color was generated. The amount of color generated by
enzymatic cleavage was proportionate to the amount of endotoxin
in the sample, and could be measured using a spectrophotometer.
Generation of color development, rather than a clot or turbidity, made
it easier to test samples that were not colorless.
In one word: sensitivity. From blood coagulation systems to cellular
signaling systems, there are a number of cascading enzymatic systems
whose mechanisms have been elucidated. Among these systems, the
horseshoe crab coagulation system, mediated by bacterial endotoxins,
is unique. Through its distinct biological amplification facility, it can
detect bacterial endotoxin concentrations to less than 1 part per trillion.