Citoxlab Newsletter – September 2017

Citoxlab North America acquires new facility in Laval/Montreal to meet the growing demand for laboratories services

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As part of our strategy to grow our global laboratory services, we are very pleased to announce the acquisition of a building that is beside our North American site in Laval (Montreal), Canada. This newly acquired ultra-modern facility is a turn-key, purpose-built, 20,000 sq ft laboratory and office building that will be used as an integral part of the planned growth of our laboratory services in support of both non-clinical and clinical research programs. Our wide range of service offerings include small and large molecule bioanalysis by LC-MS/MS and ligand binding assays: flow cytometry, biomarkers, biodistribution by qPCR, and immunology including anti-drug antibody, neutralizing antibody, immunophenotyping, ELISPOT.

“The relocation of some of our laboratory operations into this new building will free-up areas in the main building allowing us to increase our animal room capacity, resulting in shorter lead-in times for our clients. “The availability of this building literally next door to our site in Laval, was an unexpected but excellent opportunity. I am very satisfied that our fast decision-making allowed CIToxLAB to become the owner in this high-potential suburb of Montreal, where competition for this type of real estate is very high”, concluded Jean-François Le Bigot, Chairman and CEO of Citoxlab.

The obese Göttingen Minipig model

bulles NL 49_5The Citoxlab team routinely performs studies involving minipigs of various ages and sizes – from neonatal at 300 g to obese adult minipigs at 100-130 kg – each of which requires special knowledge and technical approaches.

Obesity is a growing worldwide health issue associated with an increased risk of co-morbidities such as cardiovascular disease and type II diabetes. The minipig is disposed to obesity and closely resembles humans in many features of its anatomy, physiology, biochemistry and lifestyle, which is an advantage in preclinical studies of obesity and associated diseases.

In the development of an obese large animal model, Göttingen minipigs are ovariectomised to avoid hormone-induced appetite fluctuations. The animals are fed ad libitum Altromin 9023 or a high-fat diet (Porcine G + 10% fat and 15% sucrose) in specialised feed dispensers, which monitored individual food intake. Fluctuations in food intake are noted in some animals, primarily in connection with the commencement of ad libitum feeding, resulting in a period of excessive food intake (up to 2 kg per day). The pelleted diet occasionally caused constipation and the formation of crystallised urine, which emphasises the importance of having adequate access to water. In addition, autoclaved grass-seed straw was provided to stimulate GI motility and prevent constipation and gastric ulcers caused by high food intake. Animals on ad libitum HFD gain weight very fast and tend to become too obese, which is generally not recommended.

For housing, extra large pens (3 m2/pig > 50 kg and 4 m2/pig > 100 kg) are required. In addition, water cups are enlarged to allow access for the animals as they have larger heads due to excessive cheek fat. Ample bedding is important as the obese animals will not display normal minipig behaviour or activity levels but rather spend most of the time lying down. Group housing may be attempted; the option of single housing should always be available in case of sick animals or antagonistic behaviour in the group, however.

Daily exercise is important in maintaining the health and well-being of obese minipigs. They are exercised in small groups whenever possible, and the animals are encouraged to move around during the sessions. No clear relation are observed between the willingness to move and the weight of the animals.

Lameness is observed occasionally, but is not necessarily related to the weight of the animal but more to a deviating leg composition (carpus valgus). In addition, the mobility of animals with a longer body appeared to be less affected by obesity. Thus, for obesity studies it is recommended to select animals with an appropriate body conformation.

For study-related procedures and general maintenance, there are important points to consider. Specialised equipment should be available for the weighing and moving of obese animals. A large scale is utilised and a patient lift and slings are necessary for moving anaesthetised animals for hoof trimming and other procedures. Blood sampling could not be performed by vein puncture due to the excess amount of fat in the neck region and the difficulties of safe handling and restraint of these large animals. Thus, blood is sampled via catheters inserted into the auricular vein or via an implanted vascular access port (VAP). For the implantation of the catheters, the animals are anaesthetised. The procedures required careful monitoring, as the animals are slow to recover from the anaesthesia.

In conclusion, due to the many similarities between humans and minipigs and the disposition of the minipig to obesity, this animal is considered a relevant model for obesity and obesity-related health issues. There are, however, many points to consider when developing and dealing with this model. These include the selection of animals with appropriate leg and body conformation, specialised equipment and housing and an understanding of the extra requirements of these animals.

Synthetic bone graft and bone substitute development programs: the rabbit Posterolateral Fusion (PLF) model


Autologous iliac crest bone grafting (ICBG) has represented the gold standard in orthopedics and craniomaxillofacial surgery for decades in support of implant osseointegration and surgical reconstruction. The last 20 years have seen biomaterials such as bone substitutes and synthetic bone grafts increasingly become the alternative, mainly because of the limited availability of autogenous bone grafts, prolonged surgical time and increased risk of blood loss, infections and morbidity of bone donor sites, and accumulating post-operative complications.

In order to give the best support to the bone substitutes development, and to demonstrate their safety and efficacy for regulatory submission, AccelLAB has deployed a program that includes diverse models such as long-bone critical size defects, segmental defects, craniomaxillofacial defects, intervertebral cage fusion and posterolateral (PLF) intertransverse process spine fusion models.

The latter rabbit PLF model involves the insertion of bone substitutes in the form of putty, stripes or granules, between transverse processes of adjacent vertebrae on both sides of the rabbit lumbar spine. Subsequently, the biomaterial’s capacity to promote bone bridging between transverse processes can be evaluated over time using radiography, micro computed tomography and non-decalcified bone histopathology.

AccelLAB’s latest PLF model, which was presented at the 2017 Annual Meeting of the US Society for Biomaterials, now allows for a GLP-compliant evaluation of the bone bridging and bone fusion potential of synthetic bone substitutes. With a rabbit PLF standard currently being developed by an ASTM1 working group for regulatory testing, AcceLAB is able to support the increasing demand of medtech/biotech industry for this posterolateral model.

Gastrointestinal function: Motility and Motor Migrating Complex (MMC) evaluations across models

bulles NL 49_7Drug-induced effects on gastrointestinal (GI) motility are observed with a number of approved drugs, however a relatively narrow range of non-clinical assays is available to assess GI function in drug development. Digital fluoroscopy imaging is considered a gold standard for sequential GI motility assessments. Citoxlab has recently developed a GLP compliant novel application using high definition fluoroscopic video imaging to evaluate esophageal, gastric and intestinal motility in various models using co-administration of approved radiology markers. This technology allows for GI segment specific investigations in support of drug development from discovery to regulatory studies. In addition to motility, our team has established approaches to quantify gastric and intestinal pH which may be critical to a number of oral drug delivery systems. Recently, our team has worked on the enzymatic characterization of intestinal concentrations of exocrine pancreas secretions to assist with the development of drug delivery systems in non-clinical models. Our historical data with positive and negative control agents can enhance the design of GI functional studies, but can also serve to solidify data interpretation through extensive model characterization. In addition to fluoroscopy imaging, Citoxlab has built a robust historical database with conventional GI assessment models such as the charcoal propulsion test.

Positioning of mass spectrometry and immunoassays for large molecules bioanalysis

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LC-MS–based quantitative assays are an attractive complement and potential alternative to immunoassays for large molecules. Mass spectrometry is an extraordinarily sensitive and selective technique, capable of detecting femtomoles of analytes differing in mass by fractions of a Dalton.

This is reflected in an FDA guidance, that states, “When possible, Ligand Binding Assays (LBA) should be compared with a validated reference method (such as LC-MS) using incurred samples and predetermined criteria to assess the accuracy of LBA method” (1).

Compared to LBA, Mass spectrometry detection of proteins has molecular specificity, low detection limits and a wide dynamic range. It can be performed in different ways, depending on the objective:

Top-down intact analysis – maintains protein integrity, allowing the detection of degradation products, sequence variants and combinations of post-translational modifications. This approach can be combined with highly-selective affinity purification before mass spectrometry analysis for the simultaneous qualitative and quantitative analysis of biomolecules in a single assay. This includes proteins, peptides, antibodies, antibody-drug conjugates (ADCs) and their modified forms. This approach is based on High Resolution Mass Spectrometry (HRMS) technology.

Bottom-up peptide analysis – where proteins are first proteolyzed into smaller fragments before MS interrogation to access and analyze specific peptide fragments. This method is normally performed using highly sensitive triple quadrupoles.

Should we go for triple quadrupole or HRMS?

With more than 30 MS instruments covering many different technologies including triple quadrupole (API6500 Qtrap) and HRMS (Orbitrap Q ExactiveTM) MS spectrometers, Citoxlab can support both Top-down and Bottom-up analysis.

These technologies can assist you in a wide range of applications, such as:

  • Pre-clinical and clinical analysis of biotherapeutics
  • Biomarker discovery and analysis
  • Analysis of therapeutic antibodies and antibody-drug conjugates
  • Analysis of protein variants and post-translational modifications

In the development of biologicals, these approaches are valuable for biopharmaceutical method development, biosimilar comparison to originator biopharmaceuticals, characterization of antibody–drug conjugates (ADC), and so forth.

(1) Guidance for Industry (Bioanalytical Method Validation, Biopharmaceutics)

REACH compliance 2018 deadline

bulles NL 49_8With the May 2018 REACH deadline approaching, the demand for testing is extensive and ranges from substance characterization to physicochemical, biodegradation, ecotoxicology and mammalian safety testing. It is expected that an estimated 70,000 substances will require characterization and 20,000 substances will require in vitro skin sensitization testing.

For the next few years, due to the volume of work required, there are expected to be capacity issues in areas where specialist equipment, facilities and experienced staff are limiting factors. For this reason, advance planning is needed.

There is an increasing demand for reproductive toxicology arising from the REACH deadline of May 2018 which requires OECD 421/422 studies and from ECHA Decision Letters which have requested EOGRTS (Extended One-Generation Reproductive Toxicology Studies; OECD 443) and/or developmental toxicity studies (OECD 414). ECHA have predicted approximately 200 EOGRTS studies will be required, increasing the demand at a limited choice of laboratories with experience. Citoxlab has been performing the EOGRTS since 2011 and has scientific and organizational expertise in this complex study.

Following ECHA decisions, there has also been an increased demand in repeat dose inhalation studies. Citoxlab has expanded the inhalation capacity in order to improve lead in times in this highly specialized area.

David J. Esdaile is the Director of Science and Regulatory Affairs at Citoxlab Hungary which offers a full REACH testing service including analytical support for UVCBs and difficult substances. David has been a toxicologist for over 35 years, previously working for Shell and Bayer before joining the current organization in 2004.


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European Teratology Society Meeting (ETS)
September 4-7, Budapest, Hungary

> Chemical Industry Regulations Meeting (CIR)
September 6-7, Nice, France
Booth #22

> 53rd Congress of the European Societies of Toxicology Eurotox
September 10-13, Bratislava, Slovakia
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6 scientific posters

> Citoxlab Boston Symposium: 21st Century Trends in Drug Development: Probing New Regulatory Angle
September 13, Boston, MA
Register here

> Safety Pharmacology Society Meeting (SPS)
September 24 – 27, Berlin, Germany,
Booth #103
8 scientific posters

> Preclinical Development Operations Summit 2017
October 31 – November 1, Boston, MA,
Booth #15