Abstract

Protein freeze-thawing is frequently used to stabilize and store recombinantly produced proteins after different unit operations in upstream and downstream processing. However, freeze-thawing is often accompanied by product damage and, hence, loss of product. Different effects are responsible, including cold denaturation, aggregation effects, which are caused by inhomogeneities in protein concentration, as well as pH and buffer ingredients, especially during the freeze cycle. In this study, we tested a commercially available small-scale protein freezing unit using immunoglobin G (IgG) as monoclonal antibody in a typical formulation buffer containing sodium phosphate, sodium chloride, and Tween 80. Different freezing rates were used respectively, and the product quality was tested in the frozen sample. Spatially resolved tests for protein concentration, pH, conductivity, and aggregation revealed high spatial differences in the frozen sample. Usage of slow freezing rates revealed high inhomogeneities in terms of buffer salt and protein distribution, while fast rates led to far lower spatial differences. These protein and buffer salt inhomogeneities can be reliably monitored using straight forward analytics, like conductivity and photometric total protein concentration measurements, reducing the need for HPLC analytics in screening experiments. Summarizing, fast freezing using steep rates shows promising results concerning homogeneity of the final frozen product and inhibits increased product aggregation.

Highlights

  • Antibodies are nowadays widely used for very different purposes in pharmaceutical and biotechnical industries

  • Our results show that high freezing rates are superior to low rates in terms of conformity of the sample and that these results can be transferred to larger-scale systems using the ice formation rate (IFR) as an indication factor

  • We tested the effects on the unit operation freeze-thawing onto an industrial relevant monoclonal antibody in a small-scale model

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Summary

Introduction

Antibodies are nowadays widely used for very different purposes in pharmaceutical and biotechnical industries. Negative effects of adsorption of the protein to the packaging material can be reduced by adding nonionic substances like Tween 20 or 80 to the product formulation in prefilled syringes [15]. Another strategy to minimize such interaction of the protein to interfaces is freezing of the product and minimizing the contact to other phases [11]. In phosphate-based buffers, this crystallization effect results in high deviations from the set pH upon freezing All these effects are considered to affect protein stability and promote aggregation of proteins during freezing [8,21,22,23,24,25]. Our results show that high freezing rates are superior to low rates in terms of conformity of the sample and that these results can be transferred to larger-scale systems using the ice formation rate (IFR) as an indication factor

Materials and Methods
Freeze-Thawing
Sampling
IgG Titer Determination
IgG Aggregate Determination
Conductivity Measurements
Effects on Product Quality
Findings
Conclusions
Full Text
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