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

A newly developed HPLC-MS/MS assay according to the EMA guideline for bioanalytical validation for determination of Estriol, Estradiol, and Estrone at pharm-analyt is now capable of directly (= underivatised) determining the three substances in human plasma to LLOQs of 1 pg/mL, 3 pg/mL, and 5 pg/mL respectively. Sample preparation used is quite simple which is liquid-liquid extraction; the assay was validated using ESI in negative ion mode on an API 6500 QTrap in combination with the ion mobility device Selexion. Benefits of an HPLC-MS/MS method over any kind of immuno-assay are well known and are primarily related to selectivity issues where immuno-assays often have difficulties with (key word: cross-reactivity).

With the most recent publication (Annals of Laboratory Medicine), pharm-analyt´s team has co-authored 100 publications in peer reviewed journals (Total impact factor >200)!

An inquisitive corporate culture is bound to challenge conventional approaches, leading to new ways and discoveries. If valuable for the community these ought to be published.

Most of our publications are submitted in collaboration with our partners / sponsors and related to a recent specific project or task. So “real live topics”. Some publications derive from our own research work, for instance in the field of biomarkers.
You find a comprehensive overview over all our publications under: R&D Publications or www.pharm-analyt.com/index.php/r-d.

Though self evident, it is emphasized at this point, that nothing is published without approval of the relevant stakeholders.