Our Publications & Posters

Utilizing Size Exclusion Chromatography as Universal Quality Control Analysis for Gene Therapeutics

Rebecca Hirschberger, Felix Pawel, Barbara Pfaff, Michael Rühl

Introduction:

In recent years, in vitro transcribed (IVT) messenger RNA (mRNA) gained momentum as a promising new drug class for delivering genetic information. Due to this growing interest in pharmaceutical applications, it is vital to develop reliable analytical methods for this substance class. Linear plasmids play a crucial role in IVT for the pharmaceutical production of mRNA, serving as DNA templates. For this purpose, circular plasmids with the required sequence are manufactured and converted into linear plasmids using restriction enzymes. This process results in the production of the final mRNA after successful IVT. The size exclusion chromatography (SEC) analysis presented here proves to be an effective method for the size-based separation of linear and circular plasmids, as well as for determining the length of mRNA. 

Investigation of IEX-HPLC for single base resolution sgRNA quality control 
Demonstrating a time-saving approach for good seperation in drug development 

Patrick Seyfried, Sven Warhaut, Florian Schäfer

Introduction:

CRISPR-Cas9 as a therapeutic option has proven its applicability with the market authorization of Casgevy® at the end of 2023 [1]. Companies developing single guide RNA (sgRNA) as active pharmaceutical ingredients need to assure the critical quality attributes. Among other methods, HPLC has proven to be the gold standard for high resolution separation of long oligomers [2, 3]. The sequence of the oligomer including its modifications has a strong influence on the impurity separation. This fact prevents universal methods that provide good resolution and enable the separation of molecules with truncations of only one nucleotide. Therefore, the development of individual methods is unavoidable, which makes method development for quality control time consuming, complex and expensive. 

ATR-FTIR spectroscopy: A versatile tool for incoming quality control measurements

Jens Neumann, Lena Rindermann, Jonas Watzel

Introduction:

Material identity (ID) confirmation within incoming quality control (QC) testing is routinely performed to prevent that contaminated or incorrectly labeled materials are proceeding into production. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy is a commonly used technique for this purpose in the pharmaceutical industry as a broad range of sample types like liquids, solids, and powders can be quick and easily analyzed.

Going Green - Sustainability Solutions in Oligonucleotide Manufacturing

Patrick Seyfried, Sven Warhaut, Florian Schäfer

Introduction:

Oligonucleotides have rapidly developed to become one of the most sought-after classes of drugsin the world. For more than 40 years, solid-state synthesis based on the phosphoramidite method has been the upstream process of choice, which presents several challenges in terms of sustainability, such as an unfavorable Process Mass Intensity (PMI). With increasing batch sizes and production volumes, it is essential to move towards more sustainable oligonucleotide manufacturing processes to reduce the environmental footprint.

MS/MS Quantification of therapeutic oligonucleotides via isobaric internal standard

Güngör Hanci, Michael Rühl, Christopher Gawlig

Introduction: 

Since the increasing demand for therapeutic oligonucleotides in recent years, both their qualitative and quantitative analytical characterization have become an essential analytical process. The common procedure for quantification of oligonucleotides can be carried out in multiple ways, like liquid chromatography, hybridization enzyme-linked immunosorbent assay (hELISA), and polymerase chain reaction (PCR). In particular, the quantitative analysis of oligonucleotides from biofluids and tissue is challenging. Here we describe the application of our previously published MS/MS quantification method using analytical examples based on oligonucleotide therapeutics, a 20 nt antisense oligonucleotide and a 28 nt aptamer. This quantification is performed using isobaric internal standards by multiple reaction monitoring (MRM). The standards are based on the analyte used and differ from it only by a base exchange in their sequence to create the isobaric character. Here we show that the use of isobaric internal standards, in combination with MRM analysis, yielded good accuracy, recovery, and linearity for the mid-concentration analytes (1–100 μg/mL) down to the lowest limit of quantification (LLOQ) of 20–50 ng/mL using a high-resolution qTOF system.

Comprehensive investigation of parameters influencing oligonucleotide mass spectra following IP-RP separation and negative polarity ionization

Till Wehner, Laura Steinhauer, Christopher Gawlig, Michael Rühl

Introduction: 

Oligonucleotide therapeutics are in constant development for the treatment of hereditary diseases. Companies developing such drug substances must therefore ensure the identity of these molecules, which involves the investigation and control of impurities and degradation products. Mass spectrometry has proven to be a gold standard method in determining critical quality attributes. The charge state distribution of ESI-MS spectra is highly affected by the eluent system as well as the ion source parameters. This study shows the effects of twelve eluent systems as well as the ion source parameter cone voltage, desolvation temperature, and capillary voltage, on the mass spectra of oligonucleotides ranging from 20 to 200 nucleotides.

OligoSeq: NextGen Im-/Purity Profiling Oligo Characterization by Next Generation Sequencing

Barbara Karolina Pfaff, Julius Buss, Ruven Jilly

Introduction: 

Single guide RNAs (sgRNAs) are vital for targeted therapeutics, particularly in the context of gene editing using the CRISPR-Cas9 system, and offer personalized treatments for genetic disorders, cancer, and viral infections. Therefore, quality control is crucial. Next generation sequencing (NGS) can provide a comprehensive view of oligonucleotide purity. An ultra-deep NGS method (at least 2 mio reads per sample), coupled with a bioinformatic evaluation tool, allows the characterization of sequence-based contaminants and variants. A customized pipeline solution called „Oligo Impurity Profiling Analysis“ was developed in collaboration with ecSeq Bioinformatics GmbH. This approach classifies oligonucleotide composition based on truncations, extensions, and sequence variants. The results are presented in an interactive format, providing detailed insights into sequence and length variants. The NextGen Im-/Purity Profiling approach ensures accurate assessment of synthetic oligonucleotide safety for therapeutic use.

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In vitro single guide RNA activity assay in regulated environment for quality control purposes

Christian Sattler, Felix Bade, Aref Shahnazari, Andela Juric, Julia Kraus, Michael Rühl

Introduction: 

The discovery of the bacterial immune response to phage invasion, CRISPR/Cas, has revolutionised the field of genetic research, as its use for simple and precise genetic manipulation of target cells is being applied in a variety of ways, from genome editing to therapeutic approaches [1]. The key element is the 100 nt single guide RNA (sgRNA) with its target-specific 20 nt recognition region. The sgRNA forms a complex with the Cas9 endonuclease, the ribonucleoprotein complex (RNP), and mediates the recognition of the desired DNA target site, where Cas9 performs DNA double strand cleavage. This allows it to be either re-filled with new DNA via the target-cells repair mechanism or used exclusively to cut out undesired DNA sequences. Recently, the first therapeutic based on CRISPR/Cas9 stem cell therapy received regulatory approval [2]. The high potential of this technology in the therapeutic field requires sound information of the sgRNA, e.g. sequence confirmation, purity, and activity of the sgRNA in interaction with the Cas9. While the first two are usually performed using LC-MS techniques, the determination of sgRNA-activity requires an enzymatic assay with appropriate analysis. We have developed an enzymatic in vitro activity assay, based on plasmid target-DNA and capillary electrophoresis analysis to elucidate sgRNA-dependent activity in vitro in a regulated QC-environment. The target sequence is encoded on a plasmid that is incubated with the RNP, while the RNP cleaves the target-DNA at the cleavage site into two DNA fragments. Capillary electrophoresis is used to monitor the cleavage performance respectively activity of the RNP. The generated data can be used to calculate the cutting efficiency of the RNP.

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