SPIKE RECOVERY
Endotoxin Test Concerns of Biologics
Kevin L. Williams Hospira Inc.
Limulus Amoebocyte Lysate (LAL) users are exploring regimens to study the effects of adding endotoxin to undiluted biologics in reaction to Chen’s studies (Genentech) on Low Endotoxin Recovery (LER) [1] and, moreover, in response to the addition of verbiage to the FDA Q&A Guidance [2] on establishing the “stability of assayable endotoxins content”1
in biologics. Chen’s discovery
that low levels of endotoxin added to undiluted biologics are sometimes unrecoverable by any means, leaves us to demonstrate that we do not have an endotoxin spike recovery problem with a given drug. After so many years of using Blumenthal’s in-plate spike method [3], users have grown accustomed to overlooking the potential for adverse effects from undiluted solutions. To clarify, the worry is that undiluted solutions can modify added spike (a proxy for product contamination) such that it is undetectable via LAL yet remains able to bring about fever responses in vivo. This contrasts with the recovery of in-plate spikes that are only exposed to dilute solutions of product. Chen states that LER can occur in placebo containing polysorbate with either citrate or phosphate buffer as well, thus the facts of LER are complex and still being investigated.2 out,3
Regardless of how the LER issue turns it is causing users to examine a range of spike recovery from their products.
Two broad areas of concern in the recovery of endotoxin spikes from complex protein solutions will be discussed. Both areas contain a large amount of information that must be sorted out in terms of how screening tests will be performed and the potential effects product heterogeneity may have on such testing:
1. Chen et al. spiked low levels of CSE into Biologics and could not recover them either soon after spiking (hours) or after several days (up to a week). Therefore, the converse should also be true: if low level CSE spikes are successfully recovered from undiluted drug solutions over time then we do not have either a protein binding or LER issue. Spike recovery development data is shown here for two monoclonal antibody (mAb) solutions.
2. The performance of BET (Table 5) is not conducive to preserving mAb native protein conformation and is often at odds with package insert precautions (Table 4) that center around preventing protein aggregation. endotoxin spike recovery efforts.
Such changes to proteins may exacerbate Since protein heterogeneity, especially aggregation,
is a variable factor (an inherent instability in the interaction of the molecule with minute environmental changes), preclusion by a one-time test may prove too simplistic a proposal if there are a plethora of aggregate types4 and others unavoidable5
[4, 5].
1. The full statement in Q&A question 3 is “The ability to detect endotoxins can be affected by storage and handling. Firms should establish procedures for storing and handling (which includes product mixing) samples for bacterial endotoxins analysis using laboratory data that demonstrates the stability of assayable endotoxins content. Protocols should consider the source of endotoxins used in the study, bearing in mind that purified bacterial endotoxins might react differently from native sources of endotoxins.”
2. Personal communication, LER will be the subject of a break-out session at Oct’s PDA Global Micro Conference.
Kevin Williams, currently at Hospira Inc., has 30 years’ experience in the Pharmaceutical industry specializing in endotoxin testing and control. He has written extensively on the subject of LAL
technology including authoring/editing the book “Endotoxins” (Informa Healthcare, 2007).
3. LAL users remain uninformed of many of the details of LER. Verbiage intended to preclude LER occurrence was added to the Q&A Guideline published in June of 2012.
4. See Table 1 for a list of 20 classification types [5].
5. “Aggregation can occur at any stage during manufacture, storage, distribution, or handling of products, and it results from various kinds of stress such as agitation and exposure to extremes of pH, temperature, ionic strength, or various interfaces (e.g., air– liquid interface). High protein concentrations (as in the case of some monoclonal antibody formulations) can further increase the likelihood of aggregation” [5].
4 American Pharmaceutical Review | Endotoxin Supplement 2013 induced by a variety of stressors – some avoidable
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96 |
Page 97 |
Page 98 |
Page 99 |
Page 100 |
Page 101 |
Page 102 |
Page 103 |
Page 104 |
Page 105 |
Page 106 |
Page 107 |
Page 108 |
Page 109 |
Page 110 |
Page 111 |
Page 112 |
Page 113 |
Page 114 |
Page 115 |
Page 116 |
Page 117 |
Page 118 |
Page 119 |
Page 120 |
Page 121 |
Page 122 |
Page 123 |
Page 124 |
Page 125 |
Page 126 |
Page 127 |
Page 128 |
Page 129 |
Page 130 |
Page 131 |
Page 132 |
Page 133 |
Page 134 |
Page 135 |
Page 136 |
Page 137 |
Page 138 |
Page 139 |
Page 140 |
Page 141 |
Page 142 |
Page 143 |
Page 144 |
Page 145 |
Page 146 |
Page 147 |
Page 148 |
Page 149 |
Page 150 |
Page 151 |
Page 152 |
Page 153 |
Page 154 |
Page 155 |
Page 156