(T1030-01-02) Modification of Particle and Hardware Surfaces for Efficient Biotherapeutic Method Development

Purpose: During the development and manufacturing of biopharmaceuticals, product quality attributes are monitored for product and process consistency. Method development of assays to monitor attributes of interest is often cited as an obstacle across labs due to assay sensitivity, reproducibility, and limitations associated with detector types (UV/MS). Deploying technology that can reduce challenges associated with method development would be beneficial to the biopharmaceutical industry and consequently help drive products to market faster. In this study, analyte/surface interactions were evaluated in terms of impact to the method development process. To achieve this, stationary phase and hardware surfaces of a chromatography instrument were modified to reduce nonspecific adsorption with the hypothesis being that reduced analyte/surface interaction will increase assay sensitivity through improved peak shape and chromatographic resolution as well as increase method robustness bypassing the need for surface passivation and mobile phase additives. In this work, size exclusion and ion exchange chromatography were used as common techniques deployed in the analysis of size and charge variants during the development and manufacturing of biopharmaceuticals to evaluate modified column and hardware surfaces.

Methods: NISTmAb (RM 8671) was analyzed using an isocratic gradient of 100 mM sodium phosphate pH 6.8 containing NaCl salt ranging from 0 – 200 mM. NISTmAb (RM8671) was diluted to 2 mg/mL with Milli-Q water and 1 µL injections were separated for size variants using a modified polyethylene oxide polymer stationary phase and compared to a stainless-steel diol column. The column temperature was set to 30°C with a 0.35 mL/min flowrate. The analyte was detected using a UV detector at 280 nm. Infliximab was diluted to 10 mg/mL using sterile water and was analyzed for charge variants using dual detection of optical and MS by a benchtop Q-tof. A modified SXC stationary phase column was used for separation at 40°C with a 0.1 mL/min flow rate. For optical and MS detection, mobile phases A and B contained an ammonium acetate buffer with 2% acetonitrile at acidic and basic pH. The analyte was detected using a UV detector at 280 nm and MS acquisition was performed with ESI+ and a full scan ranging from 400 – 7000 m/z.

Results: A conventional stainless-steel column and a modified polyethylene oxide polymer column were evaluated for change in HMWS % area and monomer tailing with various NaCl concentrations.  By incrementally increasing the NaCl concentrations, secondary interactions are gradually suppressed for optimal results. In triplicate injections, the modified stationary phase showed no significant change in monomer tailing from 0 – 200 mM NaCl and no significant change in HMWS % area from 50 – 200 mM NaCl. However, the stainless-steel column showed an ~35% change in monomer tailing and an ~110% change in HMWS % area at the same concentrations. The modified stationary phase yielded maximum recovery at only 50 mM NaCl concentration which required 200 mM NaCl concentration for the stainless-steel column, showing significant reduction in the need for column and system passivation. Traditionally, IEX analysis was accompanied by a salt gradient which caused MS incompatibility due to ion suppression brought on by non-volatile salts. An ammonium acetate buffer was developed to deliver a linear pH response that was compatible with optical and MS detection systems for a more complete insight into charge variant analysis. A SCX column with a modified stationary phase was coupled with the ammonium acetate buffer to yield consistent and reliable optical and MS results of an innovator and biosimilar of Infliximab. Quintuplicate injections and deconvolution of MS data showed three dominant peaks with a mass shift of +128 Da, indicating the addition of a lysine residue. Optically, these three dominant peaks showed reproducible retention times of %RSD < 1.0 demonstrating the modified column’s ability to perform consistently for charge variant separations.

Conclusion: Modification of stationary phase and hardware surfaces demonstrated reproducible and robust performance for size and charge variant analysis of biotherapeutics. By reducing the nonspecific analyte/surface interactions, methods could be quickly developed utilizing simpler mobile phases that could be utilized for optical and MS detection. This solution affords users to make informed decisions more efficiently and to advance development of products.


The modified polyethylene oxide polymer column (bottom) shows excellent monomer peak shape and HMWS recovery, even at low NaCl concentrations unlike the conventional stainless-steel column (top).


In-line pH measurements of the ammonium acetate buffer were observed to have a linear and proportional response based on gradient (left). IEX profile of the innovator using the pH gradient (right).


A mass shift of +128 Da was observed in the deconvoluted mass data indicates the addition of a lysine residue (left). Mass differences were identified as differences in glycosylation levels of the intact mAb (right).