High-throughput and high-sensitivity N-Glycan profiling: A platform for biopharmaceutical development and disease biomarker discovery.

Protein glycosylation contributes to critical biological function of glycoproteins. Glycan analysis is essential for the production of biopharmaceuticals as well as for the identification of disease biomarkers. However, glycans are highly heterogeneous, which has considerably hampered the progress of glycomics. Here, we present an improved 96-well plate format platform for streamlined glycan profiling that takes advantage of rapid glycoprotein denaturation, deglycosylation, fluorescent derivatization, and on-matrix glycan clean-up. This approach offers high sensitivity with consistent identification and quantification of diverse N-glycans across multiple samples on a high-throughput scale. We demonstrate its capability for N-glycan profiling of glycoproteins from various sources, including two recombinant monoclonal antibodies produced from Chinese Hamster Ovary cells, EG2-hFc and rituximab, polyclonal antibodies purified from human serum, and total glycoproteins from human serum. Combined with the complementary information obtained by sequential digestion from exoglycosidase arrays, this approach allows the detection and identification of multiple N-glycans in these complex biological samples. The reagents, workflow, and Hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-FLD), are simple enough to be implemented into a straightforward user-friendly setup. This improved technology provides a powerful tool in support of rapid advancement of glycan analysis for biopharmaceutical development and biomarker discovery for clinical disease diagnosis.

The role of protein hydrolysates in prolonging viability and enhancing antibody production of CHO cells.

Four independent mAb-producing CHO cell lines were grown in media supplemented with one of seven protein hydrolysates of animal and plant origin. This generated a 7x4 matrix of replicate cultures which was analysed for viable cell density and mAb productivity. In all cultures, a consistent growth rate was shown in batch culture up to 4 to 5 days. Differences between cultures appeared in the decline phase which was followed up to 7 days beyond the start of the cultures. There was a marginal but significant overall increase (x1.1) in the integral viable cell density (IVCD) in the presence of hydrolysate but a more substantial increase in the cell-specific mAb (qMab) productivity (x1.5). There were individual differences between hydrolysates in terms of enhancement of mAb productivity, the highest being a 166% increase of mAb titre (to 117 mg/L) in batch cultures of CHO-EG2 supplemented with UPcotton hydrolysate. The effect of one of the most active hydrolysates (HP7504) on antibody glycosylation was investigated. This showed no change in the predominant seven glycans produced but a significant increase in the galactosylation and sialylation of some but not all the antibodies. Overall, the animal hydrolysate, Primatone and two cotton-derived hydrolysates provided the most substantial benefit for enhanced productivity. The cotton-based hydrolysates can be viewed as valuable supplements for animal-derived component-free (ADCF) media and as a source for the investigation of chemically defined bioactive components. KEY POINTS: • Protein hydrolysates enhanced both IVCD & qMab; the effect on qMab being consistently greater. • Cotton-based hydrolysates showed high bioactivity and potential for use in serum-free media. • Enhanced galactosylation and sialylation was shown for some of the Mabs tested.

Cell Free Remodeling of Glycosylation of Antibodies.

The N-glycosylation profile of a monoclonal antibody (mAb) is a critical quality attribute in relation to its therapeutic application. The control of this profile during biomanufacture is difficult because of the multiple parameters that affect the glycosylation metabolism within the cell and the environment in which the cell is grown. One of the approaches that can be used to produce a preferred glycan profile or a single glycoform is through chemoenzymatic remodeling during the isolation of a mAb. Here we describe protocols that can be utilized to produce preferred glycoforms that include galactosylated, agalactosylated, or sialylated glycoforms following isolation of a mAb. Methods for analysis and assignment of structures of the samples following glycoengineering are also described. Chemoenzymatic modeling of mAb glycans has the potential for scale-up and to be introduced into biomanufacturing of mAbs with higher specific therapeutic activities.

Mammalian cell culture for production of recombinant proteins: A review of the critical steps in their biomanufacturing.

The manufacturing of recombinant protein is traditionally undertaken in mammalian cell culture. Today, speed, cost and safety are the primary considerations for process improvements in both upstream and downstream manufacturing. Leaders in the biopharmaceutical industry are striving for continuous improvements to increase throughput, lower costs and produce safer more efficacious drugs. This can be achieved through advances in cell line engineering, process development of cell culture, development of chemically defined media and increased emphasis on product characterization. In the first part, this review provides a historical perspective on approved biotherapeutics by regulatory bodies which pave the way for next-generation products (including gene therapy). In the second part, it focuses on the application of in vitro and in vivo cell line engineering approaches, modern process development improvements including continuous manufacturing, recent developments in media formulation, and improvements in critical quality attribute determinations for products produced predominantly in mammalian cells.

LC-MS characterization of valsartan degradation products and comparison with LC-PDA

abstract Valsartan was submitted to forced degradation under acid hydrolysis condition as prescribed by the ICH. Degraded sample aliquots were separated via HPLC using a Hypersil ODS (C18) column (250 x 4.6 mm i.d., 5 µm). Either photodiode array (PDA) detection or mass spectrometry (MS) full scan monitoring of HPLC runs were used. HPLC-PDA failed to indicate Valsartan degradation under forced acid degradation, showing an insignificant peak area variation and that Valsartan apparently remained pure. HPLC-MS using electrospray ionization (ESI) and total ionic current (TIC) monitoring did not reveal any peak variation either, but inspection of the ESI mass spectra showed the appearance of m/z 306 and m/z 352 ions for the same retention time as that of Valsartan (m/z 436). These ions were identified as being protonated molecules of two co-eluting degradation products formed by hydrolysis. These assignments were confirmed by ESI-MS/MS with direct infusion of the degraded samples. The results showed that the use of selective HPLC-MS is essential for monitoring Valsartan degradation. Efficient HPLC separation coupled to selective and structural diagnostic MS monitoring seems therefore mandatory for comprehensive drug degradation studies, particularly for new drugs and formulations, and for method development.

resumo Valsartana (VAL) foi submetida à degradação forçada em meio ácido conforme procedimento descrito no ICH. Os produtos de degradação (PDs) foram monitorados ao longo do tempo de degradação pela técnica de Cromatografia Líquida (LC) utilizando uma coluna Hypersil ODS (C18) (250 x 4,6 mm d.i., 5 µm). A detecção foi feita com dois detectores: espectrofotométrico (PDA) e espectrometria de massas (MS) por corrente iônica total. Ambas as técnicas falharam na identificação dos PDs obtidos ao longo do monitoramento, mostrando insignificantes variações na área do pico e permanecendo com pureza de pico ao longo de toda a eluição. Somente depois da avaliação por íon extraído (XIC), foi possível observar o aumento do íon m/z 306 e m/z 352 exatamente no mesmo tempo de retenção do íon molecular (m/z 436). Estes resultados mostram um caso simples e didático em que somente o uso de um método seletivo de LC-MS pode ser utilizado para monitorar produtos de degradação. Neste trabalho, é apresentado um caso real em que a separação por LC deve ser acoplada a métodos seletivos obtidos por MS, especialmente no estudo de PDs para novos fármacos, formulações e no desenvolvimento de métodos.