SPIKE RECOVERY
Table 5. BET activities known to provoke protein aggregation; advised against in package inserts
1 Shaking / vortexing
2 Addition of contaminants
Vigorous mixing is necessary to recover endotoxin but is expressly advised against in many mAbs package inserts [22].
Adding endotoxin may provoke protein aggregation via a so-called “seeding” effect: “One plausible mechanism is that aggregation is driven or catalyzed by the presence of a small amount of a contaminant” [23].
3 Immediate use 4 Use of diluents
Some mAbs are not intended to be stored after reconstitution or after opening (require immediate use).
Inserts specify diluents to use for administration. BET can be performed with any diluent(s) that aid endotoxin recovery and/or aid in overcoming interfering factors yet the use of some diluents may promote protein aggregation, especially in prolonged storage (itself often contraindicated).
of their value. The diffi culty of maintaining the natural state of biologics and the unpredictability of non-native states is hinted at below:
Recombinant human protein (rP) and glycoprotein (rGP) therapeutics are established in the clinic. However, a variety of adverse reactions are reported that may diff er between individual therapeutics, between the same therapeutic produced by diff erent companies or diff erent lots produced by the same company. Even when these parameters are controlled, there still remains the fi nal “black box”: the patient and individual disease manifestations. A feature of all rP/rGP is a potential to be immunogenic i.e., cause the generation of anti-therapeutic antibodies (ATA). Such antibodies may neutralize the therapeutic, result in enhanced clearance or precipitate severe adverse reactions. To limit the generation of ATA, an rP/rGP should, ideally, have exactly the same structure as the natural product since departure from such structural fi delity constitutes “altered-self”, and the potential to be immunogenic [15].
Summary
1. Screening for BET Spike Recovery from Undiluted Protein Solutions
A simple screen performed using low levels of CSE spikes into undiluted biologics (mAbs) has been described.
of such a test involves only a few, easily reproducible steps:
1. Establish the logistics of testing a specifi c sample based upon the (a) MVD, (b) known drug interference properties, (c) sample volume and (d) spike starting concentration.
2. Assign acceptance criteria such that a pass/fail distinction is not ambiguous.
3. Inoculate undiluted product samples with low level CSE spike (<10 EU/mL). The need for the use of >10 EU/mL solutions may arise if sample interference necessitates large dilutions.
4. Test over the course of a week (or more) for percent spike recovery. Include an in-plate PPC to gauge the on-going recovery.
5. Repeat the test to demonstrate reproducibility and consistency.
6. Ensure the key parameters of the routine test matches the test used for screening.
7. The inability to recover undiluted spike will require additional tools to address the issue (i.e. risk assessment).
8 American Pharmaceutical Review | Endotoxin Supplement 2013 In summary, the routine application
Figure 1 - Derived from Hirayama and M. Sakata’s diagram of endotoxin attachment mechanisms in protein solutions as commonly used in the removal of endotoxins via adsorbents [24].
Whereas LER is a new issue, the masking phenomenon for BET spikes into protein solutions is not new. Petsch et al. [16] used proteinase K digestion of several proteins (including human IgG) to recover endotoxin in 1998. The fact that IgG bound endotoxin shows this is not a hypothetical issue. If a screen using CSE in low levels succeeds in spike recovery from undiluted protein solutions over time, then assurance has been gained that a given drug does not have a protein binding or low endotoxin recovery (LER) issue.
2. BET at Odds with Package Insert Intent to Preserve the Native State of mAb Products
It would be a simple proposal to spike and recover endotoxin from protein solutions if mAbs were monolithic forms, however, conformations are easily changed by various BET activities that act as stressors and remain invisible to users.8,9
Package insert prohibitions safeguarding the conformation of
the proteins tested are not currently adhered to for BET because it is not clear how BET could be performed while adhering to such precautions. Neither is it clear if, when, and what aggregate states may exacerbate protein-endotoxin binding for various products.
There seems to be a
keen awareness of aggregation issues at the two ends of the spectrum (manufacturing10
analytical test performance.
and patient administration) but less so in individual For now, adding contaminants (endotoxin
spikes), storing after opening, using various diluents not specifi ed in inserts,11
and vigorous shaking is unavoidable for mAb BET testing.
BET disruption of the natural protein structure is acceptable if it does not aff ect endotoxin-protein binding (most visible here at day 7 - Table 2). This is an area that has not been a subject of study. As shown in Figure 1 there are many more variables than commonly recognized.
It is not known if
the disruption of the native states produces consistent aggregate forms (population types and proportions of various types, see Figure 2) from one BET test to the next or how these forms interact with endotoxin in its various aggregation states [20, 21].12,13
These activities (Table 5) add
a potentially confounding layer to spike studies and seem to present an opportunity to develop alternative methods of endotoxin detection that do not alter the natural state of complex proteins.
Given these various
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