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. 2021 Dec 25:4:100108.
doi: 10.1016/j.ijpx.2021.100108. eCollection 2022 Dec.

The effect of mAb and excipient cryoconcentration on long-term frozen storage stability - Part 1: Higher molecular weight species and subvisible particle formation

Oliver Bluemel  1 , Moritz Anuschek  1 , Jakob W Buecheler  2 , Georg Hoelzl  3 , Karoline Bechtold-Peters  2 , Wolfgang Friess  1
Affiliations

The effect of mAb and excipient cryoconcentration on long-term frozen storage stability - Part 1: Higher molecular weight species and subvisible particle formation

Oliver Bluemel et al. Int J Pharm X. .

Abstract

Cryoconcentration upon large-scale freezing of monoclonal antibody (mAb) solutions leads to regions of different ratios of low molecular weight excipients, like buffer species or sugars, to protein. This study focused on the impact of the buffer species to mAb ratio on aggregate formation after frozen storage at -80 °C, -20 °C, and - 10 °C after 6 weeks, 6 months, and 12 months. An optimised sample preparation was established to measure Tg' of samples with different mAb to histidine ratios via differential scanning calorimetry (DSC). After storage higher molecular weight species (HMWS) and subvisible particles (SVPs) were detected using size-exclusion chromatography (SEC) and FlowCam, respectively. For all samples, sigmoidal curves in DSC thermograms allowed to precisely determine Tg' in formulations without glass forming sugars. Storage below Tg' did not lead to mAb aggregation. Above Tg', at -20 °C and - 10 °C, small changes in mAb and buffer concentration markedly impacted stability. Samples with lower mAb concentration showed increased formation of HMWS. In contrast, higher concentrated samples led to more SVPs. A shift in the mAb to histidine ratio towards mAb significantly increased overall stability. Cryoconcentration upon large-scale freezing affects mAb stability, although relative changes compared to the initial concentration are small. Storage below Tg' completely prevents mAb aggregation and particle formation.

Keywords: Cryoconcentration; DSC, Differential scanning calorimetry; FCM, Freeze-concentrated matrix; Frozen storage; HMWS, Higher molecular weight species; HPLC, High-performance liquid chromatography; Large-scale freezing; Monoclonal antibody; PES, Polyethersulfone; SEC, Size-exclusion chromatography; SVP, Subvisible particle; Stability; Tg′, Glass transition temperature of the maximally freeze-concentrated solution; mAb, Monoclonal antibody.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Unlabelled Image
Graphical abstract
Fig. 1
Fig. 1
Changes in mAb concentration after freezing a mAb solution in histidine in a 2 L bottle. Details were published and discussed previously (Bluemel et al., 2020). The legend highlights the cryoconcentration factor, which is the ratio of the mAb concentration of the sample to the initial mAb concentration. The mAb to histidine ratios for selected samples are given in the table. Corresponding areas are highlighted for visualisation.
Fig. 2
Fig. 2
HMWS and SVPs after storage at −80 °C up to 12 months.
Fig. 3
Fig. 3
HMWS and SVPs after storage at −20 °C up to 12 months.
Fig. 4
Fig. 4
HMWS and SVPs after storage at −10 °C up to 12 months.
Fig. 5
Fig. 5
DSC thermograms of samples with different mAb to histidine ratios. The ratio of the samples was: Min His (2.58), Initial (1.61), Max mAb (1.50), Min mAb (1.16), Max His (1.00).
See this image and copyright information in PMC

References

    1. Alhalaweh A., Alzghoul A., Mahlin D., Bergström C.A.S. Physical stability of drugs after storage above and below the glass transition temperature: Relationship to glass-forming ability. Int. J. Pharmaceut. 2015;495:312–317. doi: 10.1016/j.ijpharm.2015.08.101. - DOI - PMC - PubMed
    1. Arsiccio A., Pisano R. The ice-water interface and protein stability: a review. J. Pharm. Sci. 2020;109:2116–2130. doi: 10.1016/j.xphs.2020.03.022. - DOI - PubMed
    1. Arsiccio A., McCarty J., Pisano R., Shea J.-E. Heightened cold-denaturation of proteins at the ice–water interface. J. Am. Chem. Soc. 2020;142:5722–5730. doi: 10.1021/jacs.9b13454. - DOI - PubMed
    1. Authelin J.-R., Rodrigues M.A., Tchessalov S., Singh S.K., McCoy T., Wang S., Shalaev E. Freezing of biologicals revisited: scale, stability, excipients, and degradation stresses. J. Pharm. Sci. 2020;109:44–61. doi: 10.1016/j.xphs.2019.10.062. - DOI - PubMed
    1. Barnard J.G., Singh S., Randolph T.W., Carpenter J.F. Subvisible particle counting provides a sensitive method of detecting and quantifying aggregation of monoclonal antibody caused by freeze-thawing: insights into the roles of particles in the protein aggregation pathway. J. Pharm. Sci. 2011;100:492–503. doi: 10.1002/jps.22305. - DOI - PubMed

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