David C. James

How can thermal processing modify the antigenicity of proteins

This paper is a brief review of thermally induced covalent modifications to proteins in foods, focussing mainly on the advanced glycation end‐products (AGE) of the Maillard reaction. Most foods are subjected to thermal processing, either in the home or during their production/manufacture. Thermal processing provides many beneficial effects, but also brings about major changes in allergenicity. Far from being a general way to decrease allergenic risk, thermal processing is as likely to increase allergenicity as to reduce it, through the introduction of neoantigens. These changes are highly complex and not easily predictable, but there are a number of major chemical pathways that lead to distinct patterns of modification. Perhaps the most important of these is through the reaction of protein amino groups with sugars, leading to an impressive cocktail of AGE‐modified protein derivatives. These are antigenic and many of the important neoantigens found in cooked or stored foods are probably such Maillard reaction products. A deeper understanding of thermally induced chemical changes is essential for more advanced risk assessments, more effective QC protocols, production of more relevant diagnostic allergen extracts and the development of novel protein engineering and therapeutic approaches to minimise allergenic risk.

Constraints on the transport and glycosylation of recombinant IFN-γ in Chinese hamster ovary and insect cells

In this study we compare intracellular transport and processing of a recombinant glycoprotein in mammalian and insect cells. Detailed analysis of the N-glycosylation of recombinant human IFN-gamma by matrix-assisted laser-desorption mass spectrometry showed that the protein secreted by Chinese hamster ovary and baculovirus-infected insect Sf9 cells was associated with complex sialylated or truncated tri-mannosyl core glycans, respectively. However, the intracellular proteins were predominantly associated with high-mannose type oligosaccharides (Man-6 to Man-9) in both cases, indicating that endoplasmic reticulum to cis-Golgi transport is a predominant rate-limiting step in both expression systems. In CHO cells, although there was a minor intracellular subpopulation of sialylated IFN-gamma glycoforms identical to the secreted product (therefore associated with late-Golgi compartments or secretory vesicles), no other intermediates were evident. Therefore, anterograde transport processes in the Golgi stack do not limit secretion. In Sf9 insect cells, there was no direct evidence of post-ER glycan-processing events other than core fucosylation and de-mannosylation, both of which were glycosylation site-specific. To investigate the influence of nucleotide-sugar availability on cell-specific glycosylation, the cellular content of nucleotide-sugar substrates in both mammalian and insect cells was quantitatively determined by anion-exchange HPLC. In both host cell types, UDP-hexose and UDP-N-acetylhexosamine were in greater abundance relative to other substrates. However, unlike CHO cells, sialyltransferase activity and CMP-NeuAc substrate were not present in uninfected or baculovirus-infected Sf9 cells. Similar data were obtained for other insect cell hosts, Sf21 and Ea4. We conclude that although the limitations on intracellular transport and secretion of recombinant proteins in mammalian and insect cells are similar, N-glycan processing in Sf insect cells is limited, and that genetic modification of N-glycan processing in these insect cell lines will be constrained by substrate availability to terminal galactosylation.

Monitoring recombinant human interferon-gamma N-glycosylation during perfused fluidized-bed and stirred-tank batch culture of CHO cells

Chinese hamster ovary cells producing recombinant human interferon‐γ were cultivated for 500 h attached to macroporous microcarriers in a perfused, fluidized‐bed bioreactor, reaching a maximum cell density in excess of 3 × 107 cells (mL microcarrier)−1 at a specific growth rate (μ) of 0.010 h−1. During establishment of the culture, the N‐glycosylation of secreted recombinant IFN‐γ was monitored by capillary electrophoresis of intact IFN‐γ proteins and by HPLC analysis of released N‐glycans. Rapid analysis of IFN‐γ by micellar electrokinetic capillary chromatography resolved the three glycosylation site occupancy variants of recombinant IFN‐γ (two Asn sites occupied, one Asn site occupied and nonglycosylated) in under 10 min per sample; the relative proportions of these variants remained constant during culture. Analysis of IFN‐γ by capillary isoelectric focusing resolved at least 11 differently sialylated glycoforms over a pI range of 3.4 to 6.4, enabling rapid quantitation of this important source of microheterogeneity. During perfusion culture the relative proportion of acidic IFN‐γ proteins increased after 210 h of culture, indicative of an increase in N‐glycan sialylation. This was confirmed by cation‐exchange HPLC analysis of released, fluorophore‐labeled N‐glycans, which showed an increase in the proportion of tri‐ and tetrasialylated N‐glycans associated with IFN‐γ during culture, with a concomitant decrease in the proportion of monosialylated and neutral N‐glycans. Comparative analyses of IFN‐γ produced by CHO cells in stirred‐tank culture showed that N‐glycan sialylation was stable until late in culture, when a decline in sialylation coincided with the onset of cell death and lysis. This study demonstrates that different modes of capillary electrophoresis can be employed to rapidly and quantitatively monitor the main sources of glycoprotein variation, and that the culture system and operation may influence the glycosylation of a recombinant glycoprotein.

Monitoring proteolysis of recombinant human interferon-γ during batch culture of Chinese hamster ovary cells

Proteolytic cleavage of recombinant human interferon-γ (IFN-γ) expressed in Chinese hamster ovary (CHO) cells during batch fermentation has been monitored by mass spectrometric peptide mapping. IFN-γ was purified from cell-free culture supernatant by immunoaffinity chromatography and cleaved by endoprotease Asp-N. Peptide fragments were resolved by reverse-phase HPLC and identified by a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and automated N-terminal peptide sequencing. Using this approach, a peptide was identified as the C-terminal fragment of the IFN-γ polypeptide. Analysis of this peptide by MS indicated that the recombinant IFN-γ polypeptide secreted by CHO cells was truncated by at least ten amino acids, initially at Gln133-Met134. No full length (143 amino acids) polypeptide molecules were observed at any stages of the fermentation. Additional proteolytic cleavages at basic amino acids N-terminal of Gln133 occurred during the later stages of the culture resulting in a heterogeneous IFN-γ polypeptide population with ragged C-termini.

Protein modification during antiviral heat bioprocessing.

Heat treatment is routinely used in the preparation of therapeutic protein biopharmaceuticals as a means of viral inactivation. However, in undertaking virucidal heat treatments, a balance must be found between the bioprocessing conditions, virus kill, and the maintenance of protein integrity. In this study, we utilize a simple model protein, hen egg-white lysozyme, to investigate the relationship between antiviral bioprocess conditions (protein formulation and temperature) and the extent and type of protein modification. A variety of industrially relevant wet- and dry-heat treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Although there was no evidence of lysozyme aggregation or crosslinking during any of the heat treatments, using liquid chromatography-electrospray ionization-mass spectroscopy (LC-ESI-MS) and peptide mapping we show that protein modifications do occur with increasingly harsh heat treatment. Modifications were predominantly found after wet-heat treatment, the major covalent modification of lysozyme under these conditions being glycation of Lys(97), by either glucose or fructose derived from hydrolyzed sucrose. The extent of sucrose hydrolysis was itself dependent on both the duration of heat treatment and formulation composition. Advanced glycation end products (AGEs) and additional unidentified products were also present in protein samples subjected to extended heat treatment. AGEs were derived primarily from initial glycation by fructose and not glucose. These findings have implications for the improvement of bioprocesses to ensure protein product quality.

Analysis of glycoprotein heterogeneity by capillary electrophoresis and mass spectrometry

Glycosylation is a complex posttranslational modification that can result in extensive heterogeneity for recombinant glycoproteins produced by eukaryotic systems. The carbohydrate moiety of a recombinant glycoprotein may affect the immunogenicity, half-life, bioactivity, and stability of a potential therapeutic product. Regulatory authorities such as the US Food and Drug Administration demand increasingly sophisticated carbohydrate analysis to ensure product characterization, batch-to-batch consistency, and stability.The advent of new technologies for analysis of biopolymers by capillary electrophoresis and mass spectrometry has revolutionized strategies for recombinant protein characterization. In particular, recent advances in matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry now permit relatively rapid and detaned assessment of glycoprotein and oligosaccharide structure. In this article, we describe some applications of capillary electrophoresis and mass spectrometry to monitor the glycosylation associated with a model recombinant glycoprotein, human interferon-γ.

Mechanisms of protein modification during model anti-viral heat-treatment bioprocessing of β-lactoglobulin variant A in the presence of sucrose

To ensure the safety of plasma and recombinant therapeutic proteins, heat treatment is routinely applied to these biopharmaceuticals as a means of virus inactivation. However, to maintain protein integrity during heat treatment it is necessary to use high concentrations of thermostabilizing excipients, such as sucrose, in order to prevent protein damage. In this study we describe the covalent modifications inferred to a model protein, β‐lactoglobulin A, that occur during typical and extended anti‐viral heat treatments. The chemical derivation and mechanisms by which these modifications arise are addressed. Heat treatment initiated hydrolysis of sucrose to glucose and fructose, which in turn were degraded to glyoxal. Glyoxal and the free reducing sugars reacted with free amino groups in β‐lactoglobulin A to yield Maillard glycation adducts and advanced glycation end products (AGEs). The major mechanism for AGE formation was via degradation of glucose‐derived Schiff‐base adducts. Heat treatment and glycation of β‐lactoglobulin A resulted in thiol‐disulphide interchange reactions leading to protein oligomerization. A small population of β‐lactoglobulin A non‐disulphide‐linked dimers were also observed with increasingly harsh heat treatments. These findings have implications for (i) improvements in the safety and efficacy of heat‐treated protein biopharmaceuticals and (ii) our understanding of the mechanisms of protein glycation and AGE adduct formation.

Analysis of recombinat glycoproteins by mass spectrometry.

The advent of new technologies for analysis of biopolymers by mass spectrometry has revolutionised strategies for recombinant protein characterization. The principal recent developments have been matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Using these tools, accurate molecular mass determinations can now be obtained routinely-often using minute (picomole-femtomole) quantities of protein or protein fragments. These techniques have proved indispensible for detailed characterization of the post-translational modifications of recombinant proteins produced by eukaryotic systems. Glycosylation is arguably the most important and complex of these modifications and has prompted widespread use of these new techniques. In this mini-review article I describe recent advances in the use of mass spectrometry for analysis of recombinant glycoproteins.

Protein Modifications during Antiviral Heat Bioprocessing and Subsequent Storage

Antiviral heat treatment is routinely used in the bioprocessing of therapeutic proteins as a means of reducing viral load. However, in protein formulations containing sucrose this form of bioprocessing can lead to protein modifications. Using a model protein, hen egg white lysozyme, we investigated the effects of antiviral heat treatments in the presence of sucrose on protein integrity during subsequent long‐term protein storage. Although heat treatment alone resulted in protein modification, subsequent medium‐ to long‐term storage of both lyophilized and liquid samples at room temperature or above led to further protein modifications. The majority of these modifications were due to the formation of glycation and advanced glycation end products via the reaction of reducing sugars and their autoxidation products (derived from hydrolyzed sucrose) with function groups on the protein surface. These findings have implications for the improvement of therapeutic protein bioprocessing to ensure protein product quality.

Analysis of N-Glycans by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

A naturally occurring glycoprotein is made up of a population of individual glycoforms. This molecular diversity arises from variation in oligosaccharide structures at individual glycosylation sites (microheterogeneity) and variable occupation of potential glycosylation sites (variable site occupancy or macroheterogeneity). Whilst there is no analytical method capable of preparatively resolving a glycoprotein into its discrete glycoforms, several methods have been developed in recent years to characterize, to a varying extent, populations of oligosaccharides associated with glycoproteins or glycopeptides (1). Increasingly prevalent among current analytical strategies are mass spectrometric methods. Since the early 1980s, the emergence of “soft” ionization methods for biopolymer characterization by mass spectrometry (MS) such as fast atom bombardment (FAB), electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) has provided a formidable weapon in the armoury of the analytical biochemist. Typically, an FAB ion source is accompanied by a double-sector (magnetic and electrostatic) mass analyser, ESI is usually interfaced with a quadrupole analyser and MALDI generally employs simple time-of-flight (TOF) mass analysis (2, 3, 4). All techniques are capable of low picomole to femtomole sensitivity.