www.europeanpharmaceuticalreview.

com Issue 4 · 2016

Environmental Towards a
Monitoring focus more robust
With articles from R. Vijayakumar,
Majmaah University, Tim Sandle, pharmaceutical
Bio Products Laboratory and Anastasia
Petropoulou, University Hospital Bristol’s supply chain
NHS Foundation Trust A comparison of track and trace
requirements in the EU and US
by Kathleen M. Sanzo and
Jacqueline Berman at Morgan Lewis,
and Jacob Gifford Head from
Thomas More Chambers

Spectroscopy focus
With contributions from Koen De Smet, SUPPORTING:
Tom Van Den Kerkhof and Anke Prudic from Janssen,
and University of Cambridge scientists Daniel Markl,
Michael T. Ruggiero and Axel Zeitler
3 INTRODUCTION
Contents
32 MICROBIOLOGY SERIES
Drugs research the hot Current activities of the
topic among UK politicians USP general chapters
Caroline Richards, Editor, Radhakrishna Tirumalai, PhD,
European Pharmaceutical Review United States Pharmacopeial Convention
7 FOREWORD 36 TRACK AND TRACE
EPR health-based exposure Including legal analysis from Kathleen M. Sanzo and
limits for cleaning qualification Jacqueline Berman, Morgan, Lewis & Bockius LLP,
David P. Elder, JPAG Chair and Consultant and Jacob Gifford Head, Thomas More Chambers

8 NEWS 38 WEBINAR PREVIEW

In association with Dassault Systèmes BIOVIA
10 EVENTS
11 SHOW PREVIEW
IPSE Biotech
12 REGULATORY INSIGHT
The legal framework
applicable to biosimilars in the EU
Hilary Jones, Bristows LLP

39 SPECTROSCOPY IN-DEPTH FOCUS

With contributions from Koen De Smet,
Tom Van Den Kerkhof and Anke Prudic, Janssen,
and Daniel Markl, Michael T. Ruggiero, and J. Axel Zeitler,
University of Cambridge. Turn to page 52 for
a Spectroscopy Roundtable

54 HPLC
15 ENVIRONMENTAL MONITORING
Application of LCMS in
IN-DEPTH FOCUS small-molecule drug development
With articles from R. Vijayakumar, Majmaah University, Chunang (Christine) Gu, David Russell and
Tim Sandle, Bio Products Laboratory and Anastasia Peter Yehl, Genentech
Petropoulou, University Hospital Bristol’s NHS
Foundation Trust. See page 24 for our Environmental 58 MASS SPECTROMETRY
Monitoring Roundtable
Characterising therapeutic antibodies
26 DRUG DELIVERY and ADCs using mass spectrometry
Approaches in subcutaneous Ioannis Papayannopoulos, Celldex Therapeutics
delivery of monoclonal antibodies 63 SHOW PREVIEW
Claus Geiger, Till Bussemer, Tanya Mezhebovsky
and Bernardo Perez-Ramirez, Sanofi CPhI Worldwide
COMING UP IN THE NEXT ISSUE:
Chromatography In-Depth Focus
■
■ Ingredients In-Depth Focus ■ Informatics ■ Biosimilars

Published October 2016. Don’t miss out on your copy – get your free subscription today by visiting www.europeanpharmaceuticalreview.com

VOLUME 21 ISSUE 4 2016 European Pharmaceutical Review 5

 Environmental
Monitoring

16 Bacterial endotoxin
contamination and testing
limits in ophthalmics
R. Vijayakumar, Majmaah University and
Tim Sandle, Bio Products Laboratory

20 The basics of
environmental monitoring
in aseptic units
Anastasia Petropoulou, University Hospital
Bristol’s NHS Foundation Trust

24 Environmental
Monitoring Roundtable
With Rich Quashne, WW Group Marketing
Manager, Industrial Microbiology, BD and
Patrick Hutchins, Global Product Manager
– Biotechnology, TSI Incorporated
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VOLUME 21 ISSUE 4 2016 European Pharmaceutical Review 15

 IN-DEPTH FOCUS: ENVIRONMENTAL MONITORING
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Bacterial endotoxin
contamination and testing
limits in ophthalmics
R. Vijayakumar Majmaah University  Tim Sandle Bio Products Laboratory

Endotoxin contamination in ophthalmic pharmaceuticals and medical devices presents a risk to users;
moreover, endotoxins can cause acute inflammation of the eye following ocular lens replacement surgery.
This article reviews the risks and requirements for product testing, together with current regulatory guidances.
It further analyses recall data relating to ophthalmic pharmaceuticals due to endotoxin contamination. The article
concludes by making recommendations for endotoxin control and sets out appropriate endotoxin test limits for
finished ophthalmic products.

Bacterial endotoxin is a lipopolysaccharide-protein complex that is
found in the outer cell wall of Gram-negative bacteria, including non-
infectious Gram-negative organisms. Bacterial endotoxins can exert
several physiological effects on the mammalian body (such as
endotoxemia and autoimmune disease); however, its propensity to
elicit a pyrogenic response is of greatest concern with the production of
pharmaceutical products1.
There are several potential sources of Gram-negative bacteria in
parenteral and medical device manufacturing facilities. The common
sources are the water used as the solvent or in processing; packaging
components; and the chemicals, raw materials or equipment used in
the preparation of the product. Following good manufacturing
practices, however, should sufficiently control the microbiological and
endotoxin levels in relation to each of these sources. Control is
important, since high enough endotoxin contamination in pharma-
ceutical products can cause a fever reaction in patients and, in a large
enough dose, can also cause shock and death2. Endotoxin contami-
nation affects the integrity of the products; poses an infection risk for
consumers; and can also lead to serious economic loss due to product
loss. Recently, endotoxin contamination of surgical adjuncts used in

16 European Pharmaceutical Review VOLUME 21 ISSUE 4 2016

 IN-DEPTH FOCUS: ENVIRONMENTAL MONITORING

ophthalmic surgery has become a serious concern of the United States coming from intrinsic endotoxin contamination of ophthalmic
Food and Drug Administration (FDA), largely owing to cases where vision pharmaceuticals in recent years. Kutty and her coworkers reported a
has been damaged through the use of contaminated eye products . 3
significant outbreak of TASS caused by intrinsic contamination of
a product with endotoxin from seven ophthalmology surgical centres
Concern in ophthalmic pharmaceuticals in the USA 6. The reviewers identified 112 case patients from the
Endotoxin is a potent ocular inflammatory agent and endotoxin seven centres dating from July to November 2005. Of those patients,
contamination in ophthalmic medical devices and intraocular 100 (89%) had been exposed to a single brand of balanced salt
ophthalmic products can cause ‘toxic anterior segment syndrome’ solution manufactured by Cytosol Laboratories and distributed by
(TASS)4. This is an acute inflammation of the eye following ocular lens Advanced Medical Optics, (AMO, Santa Ana, Calif.) as AMO Endosol.
replacement (cataract) surgery. Intravitreal injection of endotoxin in the A relatively high endotoxin level (0.908 EU/mL) was detected in
rabbit is a common animal model for experi- the recalled balanced salt solution, which
mental inflammation, due to physiological exceeded the allowable limit for parenteral
similarities in terms of the reaction when a Ophthalmic pharmaceuticals solutions (0.5 EU/mL).
level of endotoxin above the threshold level is manufacturers must control and monitor In 2008 another tragic outbreak was
administered into the blood stream5. Medicinal endotoxin levels in their products observed with OVD products. OVDs are visco-
products such as intraocular lenses (IOL); elastic materials used to maintain space in the
ophthalmic viscosurgical devices (OVD); ocular endotamponades and eye during surgery. Typically, they are pre-packaged in a syringe and are
balanced salt solutions that are commonly used in ophthalmic surgery, applied using a small tube. The FDA announced a Class I recall of Healon
are all at risk from endotoxin contamination. Hence, ophthalmic D (lot no. UD30654), an OVD manufactured by Advanced Medical Optics
pharmaceuticals manufacturers must control and monitor endotoxin Inc. (AMO) of Santa Ana, Calif.7 AMO received 66 subsequent adverse
levels in their products. event reports associated with the recalled products. Tests of this lot
revealed elevated levels of endotoxin, which was associated with post-
Ophthalmic pharmaceuticals recalls operative intraocular inflammation and TASS.
Devices used inside the eye, including intraocular devices and single- While only two major incidents were detected this may be an
use intraocular ophthalmic surgical instruments or accessories can underestimate. Our review was unable to recover regional level data,
potentially become contaminated with endotoxin as part of the because such data is not published in scientific journals or notified to
manufacturing, sterilisation or packaging processes. Our literature regulatory bodies via their websites.
search revealed two major outbreaks that have been reported as These concerns prompted the FDA’s Center for Devices and Radio-
logical Health (CDRH) to issue a guidance document8. The document
Table 1: Recommended endotoxin limits of ophthalmic pharmaceuticals
provides advice for endotoxin limits, together with a recommendation
Products US FDA Limit ISO Limit (Standard)
for endotoxin testing by manufacturers and other entities involved in
Anterior segment solid devices: < 0.2 EU/device < 0.5 EU/Lens
(EN-ISO-11979-8: 2011) submitting premarket applications or premarket notification
Intraocular lenses
submissions [coded 510(k)s] for different categories of intraocular
Capsular tension rings including
endocapsular rings devices to aid in the prevention of future outbreaks of TASS.8 Analysis of
Glaucoma devices – aqueous recent guidelines of endotoxin testing limits of ophthalmic
shunts, intraocular pressure-
pharmaceuticals are discussed here.
lowering implants
Phacofragmentation system – single-
use cannulated or lumened device Endotoxin limits for ophthalmic pharmaceuticals
Intraocular fluids < 0.2 EU/ml < 0.5 EU/ml Endotoxin limits of ophthalmic products set by the FDA and other
(EN-ISO-15798:2013)
Ophthalmic viscoelastic devices regulatory bodies are listed in Table 1. The FDA’s guidance has con-
(sodiumhyaluronate,
hydroxypropylmethylcellulose) cluded that existing international standards are insufficient and the
Ophthalmic endotamponades < 0.2 EU/ml < 0.5 EU/ml agency has issued a stricter standard in order to ensure that intraocular
(silicone oil, perfluoro-n-octane) (ISO 16672:2015) lenses and other intraocular ophthalmic devices are free of bacteria
Intraocular dyes – trypan blue, Not available Not available when manufactured8. Citing studies, the FDA said the International
brilliant blue G
Standards Organization (ISO) regulation for ophthalmic implants –
Retinal dyes – indocyanine green < 7.1 EU/mg* Not available
5mg/mL intraocular lenses (ISO 11979-8) limiting endotoxin to 0.5 Endotoxin
Pilocarpine nitrate 0.5% < 0.71 EU/mg NP Not available Units per millilitre in IOLs – is not sufficiently robust9. Instead, the FDA
Triamcinolone acetonide 40mg/mL < 4.4 EU/mg* Not available recommends a limit of equal to or less than 0.2 endotoxin units per
Voriconazole (1mg/mL) <1.5 EU/mg NP
Not available millilitre (EU/mL) for individual devices, regardless of whether it is
Ophthalmic sutures for cataract/ Not available Not available intended to be used in the front or back of the eye. In addition, the FDA
orbit surgery – nylon, silk, polyester, does not recognise the present endotoxin limit of OVDs specified in
polypropylene sutures
ISO 15798:2013 and ocular endotamponades specified in ISO 16672:2015
Ophthalmic surgical blades Not available Not available
(single use) (both of which have limits of not more than 0.5 EU/ml)10,11.
Eye drops Not mandatory Therefore, to minimise patient risk from the potential endotoxin
* USP standard limit; NP: non-pharmacopoeial reference, endotoxin limit for IV formulations contribution for intraocular administration, a limit of not more than

VOLUME 21 ISSUE 4 2016 European Pharmaceutical Review 17

IN-DEPTH FOCUS: ENVIRONMENTAL MONITORING
0.5 EU/dose/eye should be applied. In this case, drug products that
provide for multiple routes of administration, including intraocular
administration, would need to comply with this new limit.
However, the recent FDA document does not cover the endotoxin
limits for intraocular dyes (including indocyanin green dye, brilliant blue
G and trypan blue) and injections (such as cefotaxime, cefuroxime,
cefazolin, voriconazole and amphotericin B). In the absence of any
recommended limit, it is very important that users follow the endotoxin
limit calculation when performing the Limulus Amebocyte Lysate test,
as in USP <85>. The USP method is based on the intravenous, intra-
muscular or intrathecal dose/kg/hour (as appropriate). Given that the
eye is a much smaller and more confined space it requires a much lower
injection volume. Therefore, a limit based on
dose/kg of body weight may be inappropriate The FDA has issued a stricter
for ophthalmic injections. Here the limit standard in order to ensure that
selected must be risk-based and defensible. intraocular lenses and other intraocular
The endotoxin limit for Water-for-Injection
ophthalmic devices are free of bacteria
when manufactured
(WFI) is not more than 0.25EU/ml, following a
new amendment from the FDA for medical devices, OVD, phaco
fragmentation systems, CTRs, and glaucoma devices. Therefore, it is
important that ophthalmic manufacturers adhere to a more stringent
level of not more than 0.25EU for WFI, which adds an additional safety
factor. Due to the fact that in IOL production processing WFI plays an
important role in endotoxin removal (through rinsing and dilution),
hydrophilic lenses are packed with WFI. Notably, endotoxin contami-
nation of reusable manual ophthalmic surgical instruments, such as
PHACO probes and surgical blades, are not listed in FDA guidance.
Apart from the ophthalmic devices and intraocular injections, a
concern for endotoxin as a cause of TASS outbreak led the FDA’s Center
for Drug Evaluation and Research to consider endotoxin tests for topical
eye drops in addition to the pharmacopeial requirement for sterility.
Historically, endotoxin testing has not been considered for topical
products because the surface of the eye, such as conjunctival and
corneal epithelia, act as a natural barrier to endotoxin. We are not of
the view that endotoxin testing is necessary here, for it can be argued
that a small dose (approximately 50µL) is negligible when compared
with the proposed limit of 0.5EU/ml. Moreover, for analysis, topical
ophthalmic products are poorly suited for bacterial endotoxin tests due
to considerable interference from pH, preservatives and other
excipients12. However, we recommend that ophthalmic manufacturers
check the raw materials of WFI used for formulations of eye drops.
References
1. Mayer H, Weckesser J. The protein component of bacterial endotoxin. In: Rietschel ET,
(Ed.) Handbook of Endotoxin. Elsevier Science Publishers: NY:339; 1984
2. McCuskey R, Urbaschek R, Urbaschek B. The microcirculation during endotoxemia.
Cardiovasc Res. 1996; 32:752-763
3. Buchen SY, Calogero D, Hilmantel G, Eydelman MB. Rabbit ocular reactivity to bacterial
endotoxin contained in aqueous solution and ophthalmic viscosurgical devices. Ophthalmol.
2012; 119:e4–e10
4. Johnston J. Toxic anterior segment syndrome—More than sterility meets the eye, AORN
Journal. 2006; 84 (6): 967-975
5. Sakimoto A, Sawa M, Oshida T, et al. Minimum endotoxin concentration causing
inflammation in the anterior segment of rabbit eyes. Jpn J Ophthalmol. 2009; 53:425–32
6. Kutty PK, Forster TS, Wood-Koob C, et al. 2008. Multistate outbreak of toxic anterior
segment syndrome, 2005. J Cataract Refract Surg. 34(4):585-590
7. FDA statement at: www.fda.gov/NewsEvents/Newsroom/.../2009/ucm109057.htm
18 European Pharmaceutical Review VOLUME 21 ISSUE 4 2016
The FDA guidance also spells out the recommended testing regime.
Validation using a bacterial endotoxin test should include random
samples of lots of the devices spiked to endotoxin levels of 0.1, 0.2, and
0.5EU/mL. The percentage of endotoxin recovered from the
sterilisation process should then be calculated for each of the three
levels8. The primary endotoxin test method is the limulus amebocyte
lysate (LAL) test, as set out in the main international pharmacopeia13.
Conclusion
Bacterial endotoxin testing is a very important step in assessing
product integrity; thus ophthalmic manufacturers should be
knowledgeable about the testing methods (the Limulus Amebocyte
Lysate test) and limits for endotoxin. There is
no reference standard for some intraocular
drugs, however manufacturers should fix safe
internal limits that avoid ophthalmic inflamma-
tion. Microbiologists should be aware of the
inhibitory properties of ophthalmic dyes and
follow correct procedure to overcome this issue. This article highlights
important issues in endotoxin testing of ophthalmic dyes and
injections. However, since little data are available on the levels of
endotoxin that may damage ophthalmic tissues; some have proposed
that ophthalmic solutions and intraocular lenses should be endotoxin-
free or less than 0.2EU/ml per device. This is a position, based on the
potential risks outlined, that we support.
Dr R. Vijayakumar is an Assistant Professor of
Microbiology at the College of Science Zulfi, Majmaah
University in the Kingdom of Saudi Arabia. He has a
doctorate in microbiology from Bharathidasan University,
India. He has over 14 years’ experience in the ophthalmic
pharmaceutical industry and microbiological research.
He has written two books and published more than
15 research articles in peer-reviewed international journals. His current
research work is on ‘Biocides resistance mechanisms of environmental
and clinical isolates’.
Dr Tim Sandle is Head of Microbiology at Bio Products
Laboratory; a visiting tutor with the School of Pharmacy and
Pharmaceutical Sciences, University of Manchester; and a
long-standing committee member with the Pharmaceutical
Microbiology Interest Group (Pharmig). Dr Sandle has over
twenty-five years’ experience of microbiological research
and biopharmaceutical processing. He has written over
400 papers, book chapters and technical articles, and has presented at more
than 80 conferences and scientific meetings.
8. Endotoxin Testing Recommendations for Single-Use Intraocular Ophthalmic Devices,
Guidance for Industry and Food and Drug Administration Staff, August 17, Food and Drug
Administration, Bethesda, MD
9. ISO 11979-8:2006, Ophthalmic implants -- Intraocular lenses -- Part 8: Fundamental
requirements, International Standards Organization, Geneva
10. ISO 15798:2013 Ophthalmic implants – Ophthalmic viscosurgical device, International
Standards Organization: Geneva
11. ISO 16672:2015 Ophthalmic implants — Ocular endotamponades, International Standards
Organization: Geneva
12. Cooper JF, 2014. Endotoxin testing for topical ophthalmic products no longer required,
Special Report, Charles River Laboratories International
13. Sandle T. Bacterial Endotoxin Testing using the Limulus Amebocyte Lysate Assay.
In Kőszegi, T. and Chesca, A. (Eds.) Laboratory Techniques with Applicability in Medical
Practice, Lambert Academic Publishing. 2015; pp19-32