A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody

Cleland, Jeffrey L.; Lam, Xanthe M.; Kendrick, Brent S.; Yang, Janet Z.; Yang, Tao; Overcashier, David E.; Brooks, Dennis; Hsu, Chung Y.; Carpenter, John F.

doi:10.1002/1520-6017(200103)90:3<310::aid-jps6>3.3.co;2-i

Cited by 46 publications

(59 citation statements)

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“…Variations in random coil and b-turn structure are reflected in the region 1660-1670 cm À1 . 21 Significant differences in the aggregate species of the subclasses compared with oligomer and monomer were seen in this region. Overall, there were significant differences observed between the monomer and aggregate samples for each subclass, but the difference in the magnitude of these changes between subclasses was not clear.…”

Section: Differences In Conformational Character Between Igg1 and Iggmentioning

confidence: 95%

“…21 Both subclasses showed loss of intramolecular b-sheet structure in their aggregate species when compared with monomer, as evidenced by decreases in this1637 cm À1 band. The region 1600-1625 cm À1 reflects intermolecular b-structure 21 and both subclasses showed heterogeneous intermolecular b-sheet structure between species at this region. Variations in random coil and b-turn structure are reflected in the region 1660-1670 cm À1 .…”

Section: Differences In Conformational Character Between Igg1 and Iggmentioning

confidence: 96%

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Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

et al. 2010

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Aggregation of human therapeutic antibodies represents a significant hurdle to product development. In a test across multiple antibodies, it was observed that IgG1 antibodies aggregated less, on average, than IgG2 antibodies under physiological pH and mildly elevated temperature. This phenomenon was also observed for IgG1 and IgG2 subclasses of anti-streptavidin, which shared 95% sequence identity but varied in interchain disulfide connectivity. To investigate the structural and covalent changes associated with greater aggregation in IgG2 subclasses, soluble aggregates from the two forms of anti-streptavidin were isolated and characterized. Sedimentation velocity analytical ultracentrifugation (SV-AUC) measurements confirmed that the aggregates were present in solution, and revealed that the IgG1 aggregate was composed of a predominant species, whereas the IgG2 aggregate was heterogeneous. Tertiary structural changes accompanied antibody aggregation as evidenced by greater ANS (8-Anilino-1-naphthalene sulfonic acid) binding to the aggregates over monomer, and differences in disulfide character and tryptophan environments between monomer, oligomer and aggregate species, as observed by near-UV circular dichroism (CD). Differences between subclasses were observed in the secondary structural changes that accompanied aggregation, particularly in the intermolecular b-sheet and turn structures between the monomer and aggregate species. Free thiol determination showed 2.4-fold lower quantity of free cysteines in the IgG1 subclass, consistent with the 2.4-fold reduction in aggregation of the IgG1 form when compared with IgG2 under these conditions. These observations suggested an important role for disulfide bond formation, as well as secondary and tertiary structural transitions, during antibody aggregation. Such degradations may be minimized using appropriate formulation conditions.

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Section: Differences In Conformational Character Between Igg1 and Iggmentioning

confidence: 95%

Section: Differences In Conformational Character Between Igg1 and Iggmentioning

confidence: 96%

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

et al. 2010

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“…The structure-stabilizing effect of the saccharides reached a plateau at concentrations lower than those employed in the present study (50 mg/ml, data not shown). 21) Addition of 50 mM sodium tetraborate did not alter the BSA structure in the aqueous solution, whereas it showed a slight structure-stabilizing effect during the freeze-drying process. Co-lyophilization with sodium tetraborate (50 mM) and most of the maltooligosaccharides resulted in a BSA ahelix band broader than by maltooligosaccharide alone, indicating that the complex formation reduced the structure-stabilizing effect.…”

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confidence: 99%

Protection of Protein Secondary Structure by Saccharides of Different Molecular Weights during Freeze-Drying

Izutsu

Aoyagi

Kojima

2004

CHEMICAL & PHARMACEUTICAL BULLETIN

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Development of recombinant therapeutic proteins requires rational design of both the formulations and manufacturing processes. [1][2][3][4][5] Freeze-drying is a popular method to retain long-term stability of various proteins that are not sufficiently stable in aqueous solutions, although the stresses incurred during the freeze-drying process often induce partial unfolding and resulting protein aggregation in the rehydrated solutions. [6][7][8] Stable formulation design is required to avoid the biological activity loss and possible immunogenic effect of the structurally and/or chemically altered protein molecules.Many polyols (e.g., saccharides and sugar alcohols) protect proteins from inactivation during freeze-drying and subsequent storage. They protect native protein conformation against dehydration stresses by replacing essential water molecules through molecular interaction (e.g., hydrogen bonding) with the protein molecules.1-4,9) Some polyols also improve the physical and chemical stability of proteins during the freeze-drying process and subsequent storage by keeping the protein within a highly viscose glass-state amorphous solid with limited molecular mobility. 10,11) The choice of an appropriate saccharide and/or saccharide combination is crucial to the formulation design.1,2,12) Sucrose has been added to many freeze-drying formulations because of its potent structure-stabilizing effect and proven safety, whereas the low glass transition temperatures of the sucrose-based amorphous freeze-dried solids result in stability problems in storage at higher temperature or higher humidity. 13) Various saccharides posses different physical properties and protein-stabilizing effects.1-3) Large saccharide molecules often provide freeze-dried solids with high glass transition temperatures (T g s), which are suitable for protein storage stability. In contrast, they often show smaller effect to protect lower concentration enzymes (e.g., 1 mg/ml catalase) from inactivation during freeze-drying. 14)Protein molecules face different stresses during the freezedrying process depending on the initial concentrations, whereas the relationship between the saccharide molecular weights and the protein-stabilizing effect in freeze-drying of pharmaceutically relevant, high concentration (e.g., greater than 1 mg/ml) protein solutions has not been well elucidated.In addition to the low temperature and dehydration stresses, which are independent of the initial protein concentrations, lower concentration proteins are more likely to undergo physical stress on ice, air, and container surfaces. 3,4,15) In the present work, we used Fourier transform infrared (FT-IR) spectroscopy to study the effects of saccharides with different molecular weights on the secondary structure of proteins (BSA, ovalbumin) freeze-dried in high concentration. Addition of sodium tetraborate (Na 2 B 4 O 7 , borax) to aqueous saccharide solutions raises the "effective" saccharide molecular size through complex formation between a tetrahydroxy borate ion and one o...

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“…Protein aggregation problems are not confined to upstream bioprocessing: the techniques used to inactivate viruses during downstream processing such as exposure to detergents or extremes of pH can inadvertently damage and aggregate the protein product (Lin et al 2000). Excipients such as sugars (Cleland et al 2001) and arginine (Tsumoto et al 2005) are often used to suppress aggregate formation during protein purification and formulation. Novel fluorescence-based microtitre plate assays (Capelle et al 2007) and laser light scattering techniques (Ye 2006) will assist in the detection of protein aggregates throughout the upstream, downstream and formulation stages of bioprocessing.…”

Section: Protein Misfolding Aggregation and Methionine Oxidationmentioning

confidence: 99%

Modifications of therapeutic proteins: challenges and prospects

Jenkins

2007

Cytotechnology

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The production of therapeutic proteins is one of the fastest growing sectors of the pharmaceutical industry. However, most proteins used in drug therapy require complex post-translational modifications for efficient secretion, drug efficacy and stability. Common protein modifications include variable glycosylation, misfolding and aggregation, oxidation of methionine, deamidation of asparagine and glutamine, and proteolysis. These modifications not only pose challenges for accurate and consistent bioprocessing, but also may have consequences for the patient in that incorrect modifications or aggregation may lead to an immune response to the protein therapeutic. This review provides examples of analytical and preventative advances that have been devised to meet these challenges, and insights into how further advances can improve the efficiency and safety in manufacturing recombinant proteins.

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A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody

Cited by 46 publications

References 0 publications

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

Protection of Protein Secondary Structure by Saccharides of Different Molecular Weights during Freeze-Drying

Modifications of therapeutic proteins: challenges and prospects

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