Effect of Monosaccharides during Severe Dry Heat Treatment of Coagulation Factor VIII Concentrates

Abstract

During product development of a factor VIII concentrate (Dutch blood banks) the conversion from unsterilized to autoclaved freeze-drying buffer caused impaired product characteristics after severe dry heat treatment (80°C for 72 h). Analysis of the freeze-drying buffers showed the presence of fructose and glucose in heated buffers, resulting from hydrolysis of sucrose. The detrimental effect of glucose and fructose on solubility, yield of factor VIII and color of the heat-treated product was confirmed by freeze-drying and heating products spiked with increasing levels of these monosaccharides. The effect of the use of freeze-drying buffers autoclaved with and without sucrose was examined in two other factor VIII concentrates, S(8) and Z8 (Protein Fractionation Centre, Edinburgh, UK). If sucrose was present during autoclaving of the buffer, a slightly lower yield over freeze-drying and 80°C heat treatment was observed. Since glucose is present as a substrate in the medium for the host cells during cultivation of viruses, its potential effect on the 80°C heated product (Dutch blood banks) was examined during the validation study of the inactivation of HIV-1 and Sindbis. The cultivation cycles for the virus inolcula were simulated and residual glucose levels measured. In the supernatant medium of the host cell culture used for the propagation of Sindbis no residual glucose was found. Glucose however was found in the supernatant medium of the host cell culture for propagation of HIV-1, and therefore small molecular weight substances were removed from the actual HIV-1 inoculum by ultrafiltration. The study of the inactivation of both viruses during freeze-drying and 80°C heat treatment was subsequently carried out without complications.

Protein modification during antiviral heat bioprocessing

“…Structurally, this species can be assigned unequivocally to protein modification resulting from the condensation of 1 unit of glucose or fructose with the amino group of a lysine residue on the surface of the protein. This nonenzymatic reaction, termed protein glycation (Lapolla et al, 1996;Vasan et al, 1996), is possibly due to the hydrolysis of sucrose to yield fructose and glucose (Knevelman et al, 1994) during heat-treatment bioprocessing. Initially, the reducing sugar and primary amino group react to form a cyclized Schiff base adduct which then rearranges into the more stable ketoamine Amadori product in the case of glucose (Bucala, 1996;Zigrovic et al, 1998) or Heyns product in the case of fructose (Furth, 1997).…”

“…Recent reports suggest that hydrolysis of sucrose to fructose and glucose during autoclaving of freeze-drying buffers can have a detrimental effect on the solubility, activity, and color of factor VIII after subsequent severe dry-heat treatment (Knevelman et al, 1994). The hydrolysis may facilitate intermolecular reactions involving specific amino acids, such as the Maillard reaction, in which the amino groups of lysine residues react with reducing sugars at elevated temperatures to form a Schiff base followed by an Amadori rearrangement with polymerization (Bucala, 1996).…”

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

“…Subsequently some patients have produced neutralizing antibodies that render them refractory to further treatment [5]. Furthermore, Knevelman et al [6] have reported that the hydrolysis of sucrose to fructose and glucose during autoclaving of freeze‐drying buffers can have a detrimental effect on the activity of Factor VIII samples after subsequent dry heat treatment.…”

Evaluation of protein modification during anti‐viral heat bioprocessing by electrospray ionization mass spectrometry

“…Protein glycation has been extensively studied and the mass increase observed here (162 Da) agrees with those of previously published studies on the glycation of proteins in the presence of relatively large amounts (0.25 M) of glucose and fructose 14–17. This heat‐treatment‐induced modification is possible due to the hydrolysis of sucrose (present in the protein formulation) to yield fructose and glucose10 during bioprocessing.…”

“…Patients subsequently produce neutralizing antibodies (inhibitors) which render them refractory to further treatment 9. Furthermore, Knevelman et al have recently suggested that the hydrolysis of sucrose to fructose and glucose during autoclaving of freeze‐drying buffers can have a detrimental effect on the solubility, activity and color of factor VIII preparations after subsequent severe dry heat‐treatment 10…”