BioSpring continues to grow: new capacities for genome editing applications

Unlocking guide RNA Quality:
The Power of NGS Analysis

NGS is vital for oligo-analytics, particularly in meeting FDA recommendations for guide RNA purity analysis. Its sensitivity and accuracy are crucial for ensuring the purity and efficacy of therapeutic oligonucleotides, aligning with stringent regulatory standards. Furthermore, NGS enables detailed impurity characterization and sequence variant detection, providing researchers with deeper insights into the molecular composition and potential modifications or impurities of oligonucleotides.

Accurate NGS results depend on precise library preparation. The Illumina library preparation process for guide RNA involves critical steps that ensure reliable and reproducible sequencing data. This foundational aspect of NGS is essential for subsequent data analysis and interpretation, underscoring the importance of meticulous library preparation. At BioSpring, we introduced OligoXplore, which is a tailored bioinformatic solution designed specifically for NGS data analysis. This innovative tool facilitates the interpretation of complex sequencing data, helping researchers identify impurities and sequence variants with high precision. Effective NGS data analysis is essential for deriving meaningful insights from sequencing projects, making tools like OligoXplore invaluable.

Customized data analysis pipelines enhance the utility of NGS, providing researchers with the tools they need to address specific research challenges. These tailored bioinformatic solutions at BioSpring offer unparalleled insights into oligonucleotide purity and functionality, supporting advanced research and therapeutic development.

Dr. Pfaff's presentation at TIDES US underscored the immense potential of NGS in oligonucleotide research. Through advanced NGS techniques and bioinformatic tools, researchers can achieve a level of detail and accuracy previously unattainable. This transformative technology promises new possibilities for therapeutic development and molecular research.

Want to know more? Download our poster on NGS right here. 

Charting New Horizons in guide RNA Manufacturing

At this year’s CRISPR Medicine Conference and at TIDES US, Dr. Raoul Hennig, Head of Manufacturing Site II, delivered a captivating presentation on the evolution and efficiency of guide RNA manufacturing at BioSpring. His in-depth analysis offered attendees a comprehensive overview of our journey towards becoming the market leader in guide RNA production, focusing on driving efficiency, process scalability, and future innovations.

Over the past few years, BioSpring has made significant strides in optimizing its manufacturing processes, which have been instrumental in solidifying our leadership position. These advancements have not only enhanced the efficiency and robustness of the platform process but also ensured the high quality and reliability essential for the success of gene-editing technologies like CRISPR/Cas.

BioSpring's relentless focus on continuous improvement has led to systematic advancements from 2016 to 2020, transforming the manufacturing workflow. The introduction of Process 2.0 marked a pivotal upgrade, which translated into more consistent and reliable gRNA production. This evolution has been crucial for maintaining the high standards required in the industry.

A key highlight in recent advancements has been BioSpring's approach to process scalability. We have successfully scaled our manufacturing processes to meet the increasing demand for gudie RNA across various applications, all while maintaining the highest quality standards. This achievement underscores BioSpring's commitment to providing reliable and scalable solutions to our clients.

Our strategic vision since last year focuses on sustaining efficiency gains and further refining manufacturing processes to adapt to new challenges and opportunities. This forward-thinking approach ensures continued leadership in guide RNA manufacturing. The roadmap ahead for BioSpring is filled with exciting projects aimed at pushing the boundaries of what’s possible in guide RNA manufacturing. Future initiatives are designed to drive further efficiencies and innovations, ensuring access for our clients to the most advanced manufacturing and analytics technologies.

The comprehensive overview of the evolution and efficiency of BioSpring's guide RNA manufacturing process highlights a journey of continuous improvement and innovation. As BioSpring continues to drive the future of guide RNA manufacturing, it remains dedicated to providing updates on progress and innovations, promising an exciting horizon ahead.

Sequence confirmation of synthetic DNA exceeding 100 nucleotides using restriction enzyme mediated digestion combined with high-resolution tandem mass spectrometry

Great success for our analytical research department! In a highly acclaimed scientific paper, Dr. Michael Rühl and his team have described a new method developed in-house for the difficult challenge of sequencing long oligonucleotides. This means that even large DNA molecules can now be prepared in such a way that their sequence can be confirmed using mass spectrometry, increasing the sequence coverage of the oligonucleotides from approximately 15-25% to 100%.

Introduction:

The importance of oligonucleotide therapeutics, such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), has grown significantly, particularly for treating inherited diseases. Technologies like CRISPR-Cas9 have also highlighted the need for accurate DNA sequencing. While mass spectrometry (MS) is a standard method for analyzing RNA therapeutics, its application to long DNA molecules has been limited due to the challenges in sequencing accuracy beyond 60 nucleotides.

Methodology:

We developed a method that combines restriction enzyme digestion with high-resolution tandem mass spectrometry. The process begins with the use of common restriction enzymes to create specific digestion products from DNA molecules. These products are then analyzed using ion pair reverse phase chromatography coupled to a time-of-flight mass spectrometer. This setup allows for collision-induced dissociation (CID) for sequence analysis.

Results:

The study demonstrated that using restriction enzymes significantly increased sequence coverage from 23.3% (using direct CID-MS/MS) to 100%. The approach involved generating partial duplexes with enzyme-directing oligonucleotides, enabling site-specific cleavage by the restriction enzymes. The resulting smaller DNA fragments were easier to analyze, allowing for complete sequence verification. This method was successfully applied to single-stranded DNA molecules and a double-stranded 131 base pair DNA duplex, confirming the sequences with high accuracy.

Discussion:

We highlight the method's applicability for research, development, and quality control in regulated environments. This approach is especially valuable for confirming the sequences of synthetic DNA molecules used in therapeutics. The study notes that while traditional methods like Sanger sequencing and next-generation sequencing (NGS) are widely used, they often cannot confirm the sequence of the target molecule directly or detect certain modifications. The restriction enzyme mediated approach provides an additional layer of sequence confirmation, ensuring the highest standards of accuracy.

Conclusion:

This novel method offers a reliable and efficient solution for confirming the sequences of long synthetic DNA molecules, crucial for the development of DNA-based therapeutics. The approach's ability to provide 100% sequence coverage and its suitability for quality control makes it a valuable tool in the pharmaceutical industry. Future work may explore the applicability of this method to synthetic DNA with chemical modifications and other complex DNA structures, further enhancing its utility in genetic research and therapeutic development.

Get to know your project managers Maike Wolters Project Manager

Maike studied molecular biosciences at the Goethe University in Frankfurt, Germany. Since 2022, she is working as a project manager at BioSpring, where she supports our clients’ genome editing projects.

What do you particularly like about your work?

"What I enjoy most is the high level of communication involved and the variety of tasks offered daily. I feel like I can grow and learn in my role while contributing to the BioSpring mission. Also, it’s incredibly exciting to be involved in advancing genome editing technology with our clients and witness BioSpring’s continued growth."

How have your studies prepared you for working at BioSpring?

"Especially during my master's studies, I had the chance to attend a lot of practical courses, spend a lot of time in the lab, and engage in scientific exchanges. Beyond the technical knowledge gained, the experiences gave me a scientific understanding which is essential for the role of a project manager at BioSpring."

How many teams are involved in coordinating a GMP project?

"Coordinating a GMP project involves close collaboration across many  technical teams, including process development, quality control, quality assurance, and production. In addition, we are in regular contact with several other departments such as the accounting team and logistics team, among others."

Can you share a memorable project or accomplishment during your time here?

"A particularly memorable moment was the successful completion of my first GMP project at BioSpring. Collaborating with the client as a team and achieving our set milestones together was a rewarding experience – and it is each time again."

What do you like to do in your free time?

"To maintain a healthy life-work-balance, I enjoy various sports, from outdoorsy activities like bike tours to dancing. However, spending quality time with family and friends is what I value most!"