Purpose

For routine analysis during the manufacturing of biopharmaceuticals, HPLC systems are widely used because of their reliability and robustness. However, with regulatory bodies placing greater emphasis on method life cycle management, there is a need to explore ways to enhance the performance of HPLC instruments in terms of speed and sensitivity. In response to these regulatory pressures and to mitigate the challenges inherent in biotherapeutic analysis, a next-generation HPLC instrument platform has been developed and designed to keep pace with the progress made in biotherapeutics.

In this work, an HPLC system and column that was constructed with biocompatible materials was used for analysis of oligonucleotides and monoclonal antibodies. To evaluate the advantages that the biocompatible construction provides in the analysis of new modalities and traditional biotherapeutics, data was compared to a legacy HPLC system and evaluated on speed and recovery.

Methods                                                                

USP mAb and System Suitability standards were analyzed using an isocratic gradient of 0.20 M potassium phosphate and 0.25 M potassium chloride, pH 6.2. All standards were diluted to 10 mg/mL using the mobile phase and separated for size variants using a hydroxy terminated polyethylene oxide bonded particle on the next-generation HPLC and a stainless-steel diol column on the legacy HPLC. The column temperature was maintained at 30°C and the standards were detected using a UV detector at 280 nm.

An oligonucleotides standard and GEM91, with trace amounts of failed sequences, were analyzed through an ion-pairing RPLC method using an ethylene bridged hybrid C18 column. The ion-pairing reagent was 25 mM hexylammonium acetate, pH 7.0. The separation was done on a legacy HPLC system and then transferred to the next-generation HPLC.  The UV detector was set to 260 nm for monitoring the relevant samples and impurities.

Results 

To assess the speed and sensitivity benefits that the next-generation HPLC provides, we modernized established methods that were previously analyzed on legacy HPLC instruments. This modernization highlighted improvements in both recovery and throughput.

For the USP mAb standards, we utilized size exclusion chromatography for size variant analysis, adhering to the general requirements in USP 129. By utilizing the next-generation HPLC in conjunction with a hydroxy terminated polyethylene oxide bonded particle, the 30-minute USP method was scaled down to a 7.5-minute method while providing an approximate 10% reduction in monomer tailing. This reduction in tailing enabled integration of an embedded peak in the monomer and another LMWS, which would have otherwise remained undetected using the legacy HPLC. The reduction in monomer tailing also translated to an approximate 20% increase in resolution which enabled more precise quantitation of aggregate and fragment impurities that affect product efficacy.

Oligonucleotides have become a significant class of biotherapeutics in the new modality space but pose an analytical challenge due to their negatively charged phosphate backbone being susceptible to non-specific adsorption with metallic surfaces. This can lead to poor peak shape and recovery, making it difficult to quantitate potential impurities during manufacturing. To evaluate the biocompatible design of the next-generation HPLC, a standard containing 15, 20, 25, 30, and 35 nucleotides long oligodeoxythymidines and GEM91, a fully phosphorothioated oligonucleotide, were analyzed and compared to a legacy HPLC system. Using a ion-pairing RPLC method, a 15% increase in recovery of the oligonucleotide standard was observed in the next-generation HPLC.  The increase in recovery also enabled better resolution of impurity peaks from the main GEM91 peak for improved quantitation when compared to the legacy HPLC instrument.

Conclusions 

The next-generation HPLC with biocompatible construction demonstrated improved analysis time and sensitivity to oligonucleotides and monoclonal antibodies. The improved LC platform can be broadly deployed to analyze new modalities and traditional biotherapeutics for routine analysis during manufacturing.