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Low endotoxin recovery (LER) is a re- is caused by surfactant-generated peroxides 80 (>99% oleic acid) may prevent perox-
cently observed phenomenon referring to cause an increase in unwanted protein im- ide formation and thus increase the pho-
the inability to recover known amounts munogenicity” (4).Protein degradation has tostability of formulated solutions (11).
of endotoxin from specific stored biologi- been recognized as so severe that some (5) The high variability of polysorbate fatty
cal drug products (1). Investigators from recommend the replacement of polysor- acid content for any given formulation is
Genentech have identified two common bates as stabilizers in drug formulations. evidenced in USP/EP requirement ranges
drug excipients associated with this phe- Fatty acids are another degradation prod- and as detailed by various vendor COAs.
nomenon: polysorbate and citrate. The uct of polysorbates. A major coconstituent (e) Immunogenicity and the Pyrogen
Sigma-Aldrich catalog describes polysor- of polysorbate 20 (up to 25%) and poly- test: Many may wonder: “What is wrong
bate 20 as greater than 40% lauric acid sorbate 80 (up to 5%) is myristic acid—the with a self-depyrogenating solution?” The
and polysorbate 80 as typically 70% oleic key marker in gauging endotoxin content fear is that endotoxin may be unmasked in
acid with both having a balance of fatty via GC methods. Studies of hydroxymyris- the body as positive pyrogen data would
acid constituents. The U.S. FDA includ- tic acid as a marker for endotoxin detection suggest. The possibility remains, however,
ed a stability screen requirement in their date to the ’70s (6,7). that solutions have been inactivated in
2012 pyrogen and endotoxin guidance, (c) Mild hydrolysis: Endotoxin, biochemi- terms of endotoxin and the fever in rab-
presumably to address questions raised by cally synthesized by bacteria (seven separate bits came from immunogenicity (12) via
the Genentech study that centers on the enzymatic steps including the use of acyl- the therapeutic protein itself. It is not clear
“Stability of assayable endotoxin content.” transferases to add fatty acid acyl groups to if non-spiked solutions were tested side
The issue has come to the forefront of in- the core) (8), is a hardy molecule, removed by side with endotoxin spiked solutions
dustry in regard to the performance of the by washing/rinsing/ binding or destroyed to preclude this possibility. The LER issue
Bacterial Endotoxin Test (BET). using dry heat (250˚C for 30 min, USP). could turn out to be overstated as the cur-
Joseph Chen, PhD, and Anders Vinther, Less severe conditions, however, have also rent basis of the differential reaction said to
PhD, from Genentech, described this been found to modify the molecule so it’s exist between LAL and the pyrogen assay
phenomenon hypothetically as the forma- much less active or inactive, particularly the may be based upon borderline pyrogen
tion of a product complex that blocks the use of acids or bases with heat. Tirsoaga, et responses (all the data is not public). The
ability of factor C (the LAL biosensor) to al. (9) found even milder acidic conditions pyrogen test sets called “pyrogenic” would
bind endotoxin. In looking at the various can prompt the reaction in the presence have to be repeated on five more rabbits
substances involved including unspeci- of surfactant (SDS): “Milder hydrolysis (only day 0 and day 7 data shown) and
fied products (presumably protein and/or conditions such as pH 4.4-4.5 in sodium have at least four of eight total rabbits with
monoclonal antibodies), polysorbate and acetate buffer were shown to be efficient >0.5˚ C temperature rise to be considered
citrate, a few different potential mecha- for lipid A liberation and were usually im- pyrogenic, as per USP <151>.
nisms of action arise as listed below (a-e). proved by the addition of sodium dodecy-
Explanations that fit the Quality by De-
Users may consider various cross currents lsulfate (SDS) when the hydrolysis kinetics
sign paradigm of increasing product and
as they collectively monitor stability con- were too slow or ineffective.”
process knowledge should be explored by
ditions and consider, via a process of elim- Thus, mild acid, a surfactant/ detergent characterizing drug products, polysorbates,
ination, the LER mechanism of action. (SDS versus polysorbate) and sodium salt and degradation constituents in biologics
(a) Protein aggregation: Many proteins (acetate versus citrate) brought about sig- exhibiting the phenomenon. At this point
are known to form complexes with en- nificant hydrolysis. Mild basic hydrolysis of there are many more questions than an-
dotoxin and thus mask endotoxin in LPS has also been shown using a 1: 3 dilute swers—and answers cannot come without
solution (2). Additionally, monoclonal solution of NH4OH at room temperature widespread dissemination of details. By un-
antibodies (MAbs) are known to form for 16 hours (10). Changes in endotoxin derstanding the conditions in which LER
aggregates in aqueous solution, particu- acyl chain distribution are known to bring occurs, BET users can identify situations
larly as added surfactants (such as poly- about different biosensor responses to en- requiring greater scrutiny as to whether
sorbate) degrade spontaneously (3). dotoxin across the animal kingdom. specific drugs are affected by LER.
(b) Polysorbates: Polysorbate is used to (d) Sample hold conditions: Products References
prevent protein aggregation and loss of containing polysorbates may require the 1. J. Chen. “Low Endotoxin Recovery in
drug utility. Per Edward Maggio: “Aggre- absence of oxygen and light to prevent Common Biologics Products.” Presented
gation, which is prevented by the addition degradation. Singh, et al. suggest that re- at the 2013 PDA Annual Meeting, Or-
of surfactants, and peroxide damage, which cently introduced ultrarefined polysorbate lando, FL, April 2013