SPIKE RECOVERY
level: consistent recovery of a very low amount, ~0.05 EU/mL, after dilution. Because the adjustment of traditional development parameters (addition of buff er to diluent) resulted in improved recovery, it suggests a conventional interference mechanism (reversible binding) at work rather than an irreversible event as associated with LER of biologics as previously encountered [1]. There remains some diminishment of spike recovery, but consider that the low levels recovered represent approximately 0.1 (1:50) to 0.05 (1:100) EU/mL, which is 0.02 to 0.01 nanograms (10 to 20 picograms) of endotoxin (by defi nition relative to EC-2). Thus the diff erence in recovery of 100% versus 70% of 10 picograms represents only a 3 picogram diff erence! Spike recovery variability may sometimes seem large in CSE recovery studies, but the standard recovery metric of 50 – 200% is relative to the sensitive levels detected. Whether 1 nanogram is called a ½ nanogram or 2 nanograms should lend credence that a two-fold error is not a large amount.6
unreasonable expectation for these kind of spike recoveries.
The success of using a dilute surfactant points to a reversible aggregation mechanism while the necessity of using an additional buff er for mAb B may suggest that degradation byproducts over time may be a secondary issue.
NOE
Some advocate the use of “natural endotoxin” (non-purifi ed) for such studies, yet NOE is likely to give both more variability and confl icting results depending upon the organism(s) chosen.
The values shown by Bower
and Tran diff ered widely among cultured bacteria (they recovered <50 to >58,000 EU/mL) [8]. NOE is not really an entity that can be readily produced in the lab as it would, if natural, consist of a mixture of water-borne bacteria and cannot be ordered from ATCC7
other than as bacterial isolates (often
enterics) grown on enriched media. Many users have seen solutions of killed Gram negative bacteria increase in potency over time, presumably from the continued release of bound LPS from cellular membrane debris as solutions are vortexed over time. This could have the eff ect of “padding” the recovery of NOE spike results. Employing user-prepared NOE would make standardizing such testing very diffi cult.
2. BET at Odds with Package Insert Intent to Preserve the Native State of mAb Products
MAb Package Inserts (Table 4)
The delicate nature of mAbs as far as the prevention of protein aggregate formation is concerned is an important issue [9]. The package inserts of most mAbs detail the very specifi c conditions required to avoid aggregate formation during administration. Protein aggregate formation is an ever- present concern with mAbs during manufacture, packaging and right up to the point of injection or infusion into the patient. Formulations and containers, even container-closure coatings (i.e. silicone), have all been carefully selected to prevent aggregation [10]. Protein aggregation diminishes protein potency, can bring with it patient immunogenicity, the potential for hyperreactivity, and may be a factor in endotoxin binding of added spikes. After Herculean eff orts by manufacturers to produce and preserve the specifi c conformation of these complex molecules, the fi rst thing BET users do is disrupt the native structure. Granted, users need not adhere to package insert recommendations for reconstituting and testing products for BET (the samples are not going into a patient), yet if one is trying to recover endotoxin bound to protein then a method likely
Table 4. Commercial mAb package insert quotes on product reconstitution and dilution for administration.
(------------------®) WHICH HAS BEEN RECONSTITUTED WITH SWFI MUST BE USED IMMEDIATELY AND ANY UNUSED PORTION DISCARDED. USE OF OTHER RECONSTITUTION DILUENTS SHOULD BE AVOIDED... DO NOT SHAKE.
...the vials after reconstitution should be used immediately, not re-entered or stored. The diluent to be used for reconstitution is 10 mL of Sterile Water for Injection, USP. The total dose of the reconstituted product must be further diluted to 250 mL with 0.9% Sodium Chloride Injection, USP. Infusion should begin within 3 hours of preparation... DO NOT SHAKE.
The state of the art is such that an average of >70% is not an
Carefully inspect the solution in the (------------------®) Pen or prefilled syringe for particulate matter and discoloration prior to subcutaneous administration. If particulates and discolorations are noted, do not use the product. (------------------®) does not contain preservatives; therefore, discard unused portions of drug remaining from the syringe.
Multiple use vials: (------------------®) should be reconstituted aseptically with 1 mL of supplied Sterile Bacteriostatic Water for Injection, USP (0.9% benzyl alcohol) giving a solution of 1.0 mL containing 25 mg of (------------------®).
(------------------®) a humanized monoclonal antibody... Do not shake the content of the vial. A vigorous shaking could denaturalize the protein and affect the biological activity of the product.
Parenteral drug products should be inspected visually for particulate matter and discoloration prior to administration, whenever solution and container permit. Withdraw necessary amount of (------------------®) and dilute in a total volume of 100 mL of 0.9% Sodium Chloride Injection, USP. Discard any unused portion left in a vial, as the product contains no preservatives. DO NOT ADMINISTER OR MIX WITH DEXTROSE SOLUTION.
• DO NOT DILUTE THE PRODUCT. • DO NOT SHAKE OR VIGOROUSLY AGITATE THE VIAL. • Parenteral drug products should be inspected visually for particulate matter and discoloration prior to administration. Do not use any vials exhibiting particulate matter or discoloration.
(------------------®) may be sensitive to shear-induced stress, e.g. agitation or rapid expulsion from a syringe. DO NOT SHAKE.
Product names redacted
to cause protein aggregation may not be the best approach. Similarly, generating a form of the drug that diff ers from that used to generate other supporting analytical data (such as particulate and potency testing) seems undesirable [11]. How users could maintain the natural state of the protein while performing BET activities presents an interesting issue for future consideration.
Unknown Eff ects of Aggregates on BET (Table 5)
MAbs are a special class of molecule that have FDA-mandated testing for immunogenicity [12, 13]. If mAb protein aggregation is a factor in endotoxin-protein binding, and if mAbs are as prone to protein aggregation due to individual sample treatment as inserts suggest, then a successful study today may not preclude subsequent batches from encountering protein aggregation and thus changing the spike recovery matrix [14]. Arguably, the presence of various aggregate population types batch to batch could call into question the ability of a one-time test to preclude all spike loss scenarios. On the other hand, successful spike recovery via a simple screen may be the best precaution currently available. stated, adding complexity around such studies adds no further guarantee
Simply
6. EU is defi ned as 0.2 nanogram of EC-2 (1/5 the 1 ng/kg established as the threshold pyrogenic response) [7] 7. American Type Culture Collection
7
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