We are proud and excited to announce, that pharm-analyt has acquired another last generation mass-spec-system QTRAP® 6500 from AB Sciex!

pharm-analyt was nominated for the innovation award “Kreativ in die Zukunft” ("Creatively Into the Future") in the category “Technical Innovation”.

The award is sponsered by the Austrian Economic Chamber.  The final prize will be awarded on June 10^th in St. Pölten.

pharm-analyt discovered and validated specific substances (Small Molecules) in plasma that indicate particular genetic disorders with remarkably high specificity and sensitivity.

Three of these biomarkers were submitted and nominated for this innovation award: For Gaucher’s Disease, Niemann-Pick Type C and Metachromatic Leucodystrophy.
All three have orphan status with an incidence rate of 1:20,000, 1:150,000 and 1:50,000, respectively.

pharm-analyt acquired broad experience and knowledge over the last years in the area of lysosomal storage diseases.

After identifying and validating biomarkers for several lysosomal storage diseases (Gaucher, Fabry, NPC, MLD), pharm-analyt now identified  another biomarker in this field:

A diagnostic marker for Krabbe Disease.

About Krabbe disease (quote from U.S. National Library of Medicine)

Krabbe disease (also called globoid cell leukodystrophy) is a degenerative disorder that affects the nervous system. It is caused by the shortage (deficiency) of an enzyme called galactosylceramidase. This enzyme deficiency impairs the growth and maintenance of myelin, the protective covering around certain nerve cells that ensures the rapid transmission of nerve impulses. Krabbe disease is part of a group of disorders known as leukodystrophies, which result from the loss of myelin (demyelination). This disorder is also characterized by the abnormal presence of globoid cells, which are globe-shaped cells that usually have more than one nucleus.

The symptoms of Krabbe disease usually begin before the age of 1 year (the infantile form). Initial signs and symptoms typically include irritability, muscle weakness, feeding difficulties, episodes of fever without any sign of infection, stiff posture, and slowed mental and physical development. As the disease progresses, muscles continue to weaken, affecting the infant's ability to move, chew, swallow, and breathe. Affected infants also experience vision loss and seizures.

Less commonly, onset of Krabbe disease can occur in childhood, adolescence, or adulthood (late-onset forms). Visual problems and walking difficulties are the most common initial symptoms in this form of the disorder, however, signs and symptoms vary considerably among affected individuals.

Incidence Rate:

In the United States, Krabbe disease affects about 1 in 100,000 individuals. A higher incidence (6 cases per 1,000 people) has been reported in a few isolated communities in Israel.

GMP cleaning validations are a critical part of the manufacturing process of specific galenic formulations.
In order to rule out contamination by potent APIs on working surfaces, highly sensitive analytical methods have to be established.  

pharm-analyt has validated numerous HPLC-MS/MS-methods for various APIs over the last years and has been conducting swab analyses on a continuous basis.  

pharm-analyt has been GMP certified since 2006 and GLP certified since 1992.

Please do not hesitate to contact us in regard to your cleaning validations!

Martin van Dam
Director Business Development
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+43 664 5160 842

EXPERT EXPERIENCES

The importance of PEGylated substances has been increasing over the past years, driven by the demand of improved control-release of drugs. Naturally this again has been raising the demand for quantification of respective PEGylated drugs in plasma...

pharm-analyt has developed innumerous assays over the past years for quantification of such PEGylated drugs from plasma and urine in clinical and preclinical context.

Here we want to show two examples from our daily experience analyzing PEGylated drugs.

Example A)
A relatively small PEGylated substance should be determined together with its demethylated free carboxylic acid form. The un-PEGylated substance itself had a molecular weight of about 500 Da, a 2 kDa PEG was introduced to it. An extraction method from human plasma and human urine could successfully be developed; luckily the substance itself contained a fluorophore and could therefore be determined by an HPLC-Fluorescence method with an LLOQ of 2 ng/mL in human plasma and 5 ng/mL in human urine.

The method could be successfully validated for both matrices in GLP context. Limitations were selectivity, otherwise a lower quantitation limit would have been possible. Detection limits with HPLC-MS/MS would have been lower by possibly a factor of about 10, however reproducibility was poor since internal standards available were too different from the analytes and the distribution of the 2 kDa PEG (in terms of molecular weight) introduced in the substance was not homogenous enough.

Example B)
A Method for determination of a large PEG of more than 40 000 Da with a small molecule linked to it (< 2000 Da) was successfully developed and validated in GLP context for plasma and urine of humans and animals. An extraction method could successfully be applied; quantitation is done with HPLC-MS/MS in ESI positive mode of the highly charged analyte on size exclusion columns (different materials used so far). Actual validated assays have quantitation limits of 0.1 µg/mL plasma or urine after extraction. An assay that was not fully validated in GLP context yet (but eventually will be when required) will be capable of quantifying 10 ng/mL of this quite large PEGylated substance!

Based on these examples as well as from extensive experience with other similar substances we believe that we can quantify even PEGs in the range of more than 100 000, maybe even up to 500 000 Da from biological matrices such as plasma or urine of animals and humans to possibly LLOQs in the range of about 100 ng/mL.

What needs to be borne in mind for such assays, of course, is that selectivity majorly is gained by chromatography, not by MS/MS detection. Therefore the assay may not be capable of discerning the analyte from possible metabolites or “degradation” products with small loss in molecular weight. This “problem” however is not new to people working with very large molecules (glycosylation…. for other substance classes).

These are just two examples from our daily life working with PEGylated drugs. Over the last several years pharm-analyt has continuously been solving analytical challenges of various PEGs with HPLC-MS/MS, including PEGylated peptides and PEGylated liposomes (“stealth liposomes”).

25. 03. 2014, Dr. Daniel Mascher, CSO pharm-analyt Lab. GmbH