Parallel development of chromatographic and mass-spectrometric methods for quantitative analysis of glycation on an IgG1 monoclonal antibody.

Monitoring post-translational modifications (PTMs) in biotherapeutics is of paramount importance. In pharmaceutical industry, chromatography with optical detection is the standard choice of quantitation of product related impurities; and mass spectrometry is used only for characterization. Parallel development of a boronate affinity chromatographic (BAC) and a mass spectrometric methods for quantitative measurement of glycation on a monoclonal antibody (mAb) shed light on the importance of certain characteristics of the individual methods. Non-specific interactions in BAC has to be suppressed with the so-called shielding reagent. We have found that excessive amount of shielding reagents in the chromatographic solvents may cause significant underestimation of glycation. Although contamination of the retained peak with the non-glycated isoforms in BAC is unavoidable, our work shows that it can be characterized and quantitated by mass spectrometry. It has been demonstrated that glycation can be measured by mass spectrometry at the intact protein level with an LOQ value of 3.0% and error bar of ±0.5%. The BAC and MS methods have been found to provide equivalent results. These methods have not been compared from these points of view before.

Critical evaluation of fast size exclusion chromatographic separations of protein aggregates, applying sub-2 µm particles.

A new size exclusion chromatography column packed with 1.7 µm particles and possessing 200 Å pore size has been critically evaluated for the determination of proteins and monoclonal antibody aggregates. In a first instance, the kinetic performance of this column was compared with that of a conventional column packed with 5 µm particles and with a recently launched 3 µm material, also possessing 200 Å pore size. In average, 2-5 times lower plate height were achieved on the 1.7 µm packing, compared with the conventional 5 µm particles. It was also demonstrated that elevated mobile phase temperature (up to 50 or 60 °C) allows improving the kinetic efficiency by 20-40% in size exclusion chromatography, compared to 30 °C. On the other hand, the new 3 µm material performed only slightly lower kinetic efficiency than the 1.7 µm one. When considering the upper pressure and temperature limits of these three columns, the 1.7 µm column systematically outperforms the 5 and 3 µm materials in the "practical" plate number range (N<30,000) and analysis times could be cut by 2-4 times. The column packed with 5 µm particles was only beneficial for plate counts beyond 100,000 plates, while the 3 µm packing could be considered as a good compromise between speed, efficiency and pressure. Besides the excellent kinetic performance of 1.7 µm size exclusion material under high temperature conditions, some artifacts were observed when quantifying protein aggregates. Indeed, both high pressure observed with 1.7 µm particles (shear forces, frictional heating) and elevated temperature produce some non negligible amount of on-column additional protein aggregates.

Analytical aspects of biosimilarity issues of protein drugs.

The term "biosimilar" is used to designate a follow-on biologic that meets extremely high standards for comparability or similarity to the originator biologic drug that is approved for use in the same indications. Use of biosimilar products has already decreased the cost of treatment in many regions of the world and now a regulatory pathway for approval of these products has been established both in US and in the EU. The analytical tests to demonstrate comparability and similarity of a biosimilar product to a reference drug with respect to protein content, activity, physiochemical integrity, stability, impurities and additives, as well as immunogenicity are discussed.

Efficiency of the new sub-2 µm core-shell (Kinetex™) column in practice, applied for small and large molecule separation.

At present sub-2 µm packed columns are very popular to accomplish rapid and efficient separations. Applying particles with shortened diffusion path to improve the efficiency of separation performs higher efficiency than it is possible with the totally porous particles having the same size. The advantages of sub-2 µm particles and shell particles are combined in the new Kinetex 1.7 µm particles. In this study a systematical evaluation of the efficiency and achievable analysis time obtained with 5 cm long narrow bore column packed with sub-2 µm core-shell particles (1.25 µm core diameter and 0.23 µm porous silica layer), and other type very efficient columns is presented. The efficiency of separation was investigated also for small pharmaceutical and large molecules (proteins). Van Deemter, Knox and kinetic plots are calculated. The results obtained with low molecular weight polar neutral analytes (272 g/mol, 875 g/mol), with a polypeptide (4.1 kDa) and with different sized proteins (18.8 kDa, 38.9 kDa and 66.3 kDa) are presented in this study. Moreover, particle size distribution, and average pore size (low-temperature nitrogen adsorption, LTNA) of the new very fine core-shell particles were investigated. According to this study, increased flow rates can be applied on sub-2 µm core-shell columns to accomplish very fast separations without significant loss in efficiency. The new sub-2 µm shell particles offer very high efficiency both for small and large molecule separation.

Simultaneous determination of polysorbate 20 and unbound polyethylene-glycol in protein solutions using new core-shell reversed phase column and condensation nucleation light scattering detection.

A novel fast and sensitive method has been developed for the specific simultaneous determination of polysorbate 20 (Tween 20) and unbound polyethylene-glycol (PEG) from liquid formulations in the presence of proteins and excipients. The quantitative determination is based on a fast liquid chromatographic (HPLC) separation and condensation nucleation light scattering detection (CNLSD or NQAD™). The method uses a Kinetex core-shell column (100 mm × 3 mm, 2.6 µm) and methanol-water-trifluoroacetic acid mobile phase. The rapid HPLC-CNLSD method presented here is suitable for quantifying polysorbate 20 in the range of 10-60 µg/ml and unbound PEG in the range of 2-40 µg/ml in protein solutions within good manufacturing practices (GMP) of the pharmaceutical industry.

Comparative study of new shell-type, sub-2 micron fully porous and monolith stationary phases, focusing on mass-transfer resistance.

Today sub-2 microm packed columns are very popular to conduct fast chromatographic separations. The mass-transfer resistance depends on the particle size but some practical limits exist not to reach the theoretically expected plate height and mass-transfer resistance. Another approach applies particles with shortened diffusion path to enhance the efficiency of separations. In this study a systematical evaluation of the possibilities of the separations obtained with 5 cm long narrow bore columns packed with new 2.6 microm shell particles (1.9 microm nonporous core surrounded by a 0.35 microm porous shell, Kinetex, Core-Shell), packed with other shell-type particles (Ascentis Express, Fused-Core), totally porous sub-2 microm particles and a 5 cm long narrow bore monolith column is presented. The different commercially available columns were compared by using van Deemter, Knox and kinetic plots. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Data are presented on polar neutral real-life analytes. Comparison of a low molecular weight compounds (MW=270-430) and a high molecular weight one (MW approximately 900) was conducted. This study proves that the Kinetex column packed with 2.6 microm shell particles is worthy of rivaling to sub-2 microm columns and other commercially available shell-type packings (Ascentis Express or Halo), both for small and large molecule separation. The Kinetex column offers a very flat C term. Utilizing this feature, high flow rates can be applied to accomplish very fast separations without significant loss in efficiency.

Fast and sensitive determination of Polysorbate 80 in solutions containing proteins.

A fast and sensitive method has been developed for the specific determination of Polysorbate 80 (Tween 80) in liquid formulations in the presence of proteins and excipients. The quantitative determination is based on a fast liquid chromatographic (HPLC) separation and charged aerosol detection (CAD). The method was validated using a Poroshell 300SB-C18 column packed with 5 microm shell particles (75 mm x 2.1 mm) and acetonitrile-methanol-water-trifluoroacetic acid mobile phase at a flow rate of 0.65 ml/min. The rapid LC-CAD method is suitable for quantifying Polysorbate 80 in the range of 10-60 microg/ml in protein solutions within good manufacturing practices (GMPs) of the pharmaceutical industry.

Facts and myths about columns packed with sub-3 microm and sub-2 microm particles.

Increasing the separating efficiency enhances the separation power. The most popular solution for improving chromatographic performance is to employ columns packed with small particle diameters (i.e., sub-2 microm particles) to induce a simultaneous improvement in efficiency, optimal velocity and mass transfer, albeit the cost of pressure. In this study a systematic evaluation of the possibilities and limitations of the separations obtained with 5 cm long narrow bore columns packed with 1.5-3.0 microm particles is presented. Several commercially available different sub-3 microm and sub-2 microm packed columns were evaluated by using van Deemter, Knox and kinetic plots. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Data are presented on different polar neutral real life analytes, to show that the separation time is not obviously shorter if the particle size is reduced. Comparison of low-molecular weight compounds (one steroid and one non-steroid hormone, with molecular weights lower than 500) and a high-molecular weight one (MW approximately 1000) was conducted. Same efficiency can be achieved with columns packed with 1.9-2.1 microm particles as with smaller particles. The column packed with 3 microm particles had the lowest reduced plate height minimum (h=2.2) while the column with the smallest particles (1.5 microm) gave the highest reduced plate height minimum (h approximately 3.0). According to this study, the theoretically expected efficiency of very fine particles (diameter <2 microm) used in practice today is compromised. Investigation of this phenomenon is presented.

Rapid high performance liquid chromatography method development with high prediction accuracy, using 5cm long narrow bore columns packed with sub-2microm particles and Design Space computer modeling.

Many different strategies of reversed phase high performance liquid chromatographic (RP-HPLC) method development are used today. This paper describes a strategy for the systematic development of ultrahigh-pressure liquid chromatographic (UHPLC or UPLC) methods using 5cmx2.1mm columns packed with sub-2microm particles and computer simulation (DryLab((R)) package). Data for the accuracy of computer modeling in the Design Space under ultrahigh-pressure conditions are reported. An acceptable accuracy for these predictions of the computer models is presented. This work illustrates a method development strategy, focusing on time reduction up to a factor 3-5, compared to the conventional HPLC method development and exhibits parts of the Design Space elaboration as requested by the FDA and ICH Q8R1. Furthermore this paper demonstrates the accuracy of retention time prediction at elevated pressure (enhanced flow-rate) and shows that the computer-assisted simulation can be applied with sufficient precision for UHPLC applications (p>400bar). Examples of fast and effective method development in pharmaceutical analysis, both for gradient and isocratic separations are presented.

Characterization of new types of stationary phases for fast liquid chromatographic applications.

The performance of a narrow bore silica based monolith column (5 cm x 2 mm) was compared to 5 cm long narrow bore (internal diameter < or = 2.1 mm) columns, packed with shell particles (2.7 microm) and totally porous sub-2 microm particles (1.5 microm, 1.7 microm and 1.9 microm) in gradient and isocratic elution separations of steroids. The highest peak capacity could be achieved with the column packed with 1.5 microm totally porous particles. The columns packed with porous 1.7 microm and shell 2.7 microm particles showed very similar capacity. The monolith column provided the lowest capacity during gradient elution. The plate height (HETP) of the 2.7 microm Ascentis Express column was very similar to the HETP obtained with 1.5 microm and 1.7 microm totally porous particles. The Chromolith monolithic column displayed an efficiency that is comparable to that of columns packed with spherical particles having their diameter between 3 microm and 4 microm. A kinetic plot analysis is presented to compare the theoretical analysis speed of different separation media. At 200 bar, the monolith column provided the highest performance when the required plate number was higher than 5000 (N>5000), however the efficiency drifted off faster in the range of N<5000 than in the case of packed columns. If the possibility of maximum performance was utilized (1000 bar for sub-2 microm particles, 600 bar for shell particles and 200 bar for monolith column) the monolith column would provide the poorest efficiency, while the column, packed with 1.5 microm particles offered the shortest impedance time.

Validated UPLC method for the fast and sensitive determination of steroid residues in support of cleaning validation in formulation area.

An ultra performance liquid chromatographic (UPLC) method was developed for simultaneous determination of seven steroid (dienogest, finasteride, gestodene, levonorgestrel, estradiol, ethinylestradiol, and norethisterone acetate) active pharmaceutical ingredient (API) residues. A new, generic method is presented, with which it is possible to verify the cleaning process of a steroid producing equipment line used for the production of various pharmaceuticals. The UPLC method was validated using an UPLC BEH C18 column with a particle size of 1.7 microm (50 mm x 2.1 mm) and acetonitrile-water (48:52, v/v) as mobile phase at a flow rate of 0.55 ml/min. Method development and method validation for cleaning control analysis are described. The rapid UPLC method is suitable for cleaning control assays within good manufacturing practices (GMP) of the pharmaceutical industry.

Shell and small particles; evaluation of new column technology.

The performance of 5 cm long columns packed with shell particles was compared to totally porous sub-2 microm particles in gradient and isocratic elution separations of hormones (dienogest, finasteride, gestodene, levonorgestrel, estradiol, ethinylestradiol, noretistherone acetate, bicalutamide and tibolone). Peak capacities around 140-150 could be achieved in 25 min with the 5 cm long columns. The Ascentis Express column (packed with 2.7 microm shell particles) showed similar efficiency to sub-2 microm particles under gradient conditions. Applying isocratic separation, the column of 2.7 microm shell particles had a reduced plate height minimum of approximately h=1.6. It was much smaller than obtained with totally porous particles (h approximately = 2.8). The impedance time also proved more favorable with 2.7 microm shell particles than with totally porous particles. The influence of extra-column volume on column efficiency was investigated. The extra-column dispersion of the chromatographic system may cause a shift of the HETP curves.

Content uniformity and assay requirements in current regulations.

The acceptance of a tablet batch is based both on the content uniformity test and on the assay. It is shown that these two characteristics are not independent, and the acceptance criteria for them are not even consistent. For content uniformity range three methods of calculation are compared: the present European Pharmacopoeia method, a tolerance range method with improved k tolerance factor and a one-way random effects analysis of variance model. To resolve the inconsistency several options are discussed: applying the holistic content uniformity range alone; using content uniformity standard deviation and assay mean simultaneously or applying a criterion based on Taguchi's quadratic loss function.