Impact of Freeze -Thaw on monoclonal Antibody solutions

Monoclonal Antibodies – or mAbs for short

Since many of these mAbs require refrigeration we had used dynamic light scattering (with a Zetasizer) to show the formation of aggregates after freeze-thaw cycles (for details see application note “Effect of storage conditions on Immunoglobulin“). These could be seen both in the increased polydispersity index (PDI) and the appearance of a large-size aggregate peak in the intensity size distribution as shown to the right. The area under the peak gives an indication of the presence of the large aggregates but is not suited for quantitative %aggregation by mass studies, where chromatography (like the OMNISEC) is superior.

In a more recent article from K. Desai et al., “Impact of Manufacturing-Scale Freeze-Thaw Conditions on a mAb Solution,” BioPharm International 30 (2) February 2017. showed some interesting results, where size exclusion chromatography (SEC) and micro-flow imaging (MFI) were the key techniques to determine % aggregation and subvisible particle number concentration, respectively. Their formulation contained IgG1 at 64mg/mL in a 10mM buffer with added surfactant, a disaccharide as cryoprotectant and an amino acid (Glycine). They looked at the effect of container volume and speed of freezing and thawing.

Here is what I found especially interesting:

1. More freeze-thaw cycles produce more aggregates, both in terms of quantifiable SEC and countable particulates
2. Small scale containers showed more aggregates
3. Slow thawing produced more aggregates than fast thawing
4. Fast freezing produced more aggregates than slow freezing, but fast freeze followed by fast thaw was found as optimal

The study also included differential scanning calorimetry (DSC) data to support the hypothesis that slow thawing could lead to more glycine (eutectic) crystallization and melt. Interestingly, we actually have quite a few of the techniques available as part of our Malvern-Panalytical’s biologic characterization tools.

To find out more about the techniques mentioned in the article and for related notes of interest:

• White Paper “A guide to protein stability measurement platforms“
• White Paper “Developing a bioformulation stability profile“
• Application Note “Comparing DLS and AUC for IgG4“
• Blog “Three ways to characterize a mAb with the ZMV“
• Blog “The chromatogram series: Fab“

Feel free to contact us if you’d like to get extra insights into your monoclonal antibody, biosimilar, or biotherapeutic. We can run samples.

Previously

• Can I use the Zetasizer for studying…?
• New Nanosizer users – quick guide to perfection
• Elucidation of aggregation kinetics with DLS

If you have any questions, please email me at ulf.nobbmann@malvern.com. Thanks! While opinions expressed are generally those of the author, some parts may have been modified by our editorial team.