Impact of iron raw materials and their impurities on CHO metabolism and recombinant protein product quality

Abstract

Cell culture medium (CCM) composition affects cell growth and critical quality attributes (CQAs) of monoclonal antibodies (mAbs) and recombinant proteins. One essential compound needed within the medium is iron because of its central role in many cellular processes. However, iron is also participating in Fenton chemistry leading to the formation of reactive oxygen species (ROS) causing cellular damage. Therefore, this study sought to investigate the impact of iron in CCM on Chinese hamster ovary (CHO) cell line performance, and CQAs of different recombinant proteins. Addition of either ferric ammonium citrate (FAC) or ferric citrate (FC) into CCM revealed major differences within cell line performance and glycosylation pattern, whereby ammonium was not involved in the observed differences. Inductively coupled plasma mass spectrometry (ICP-MS) analysis identified varying levels of impurities present within these iron sources, and manganese impurity rather than iron was proven to be the root cause for increased cell growth, titer, and prolonged viability, as well as altered glycosylation levels. Contrary effects on cell performance and protein glycosylation were observed for manganese and iron. The use of low impurity iron raw material is therefore crucial to control the effect of iron and manganese independently and to support and guarantee consistent and reproducible cell culture processes.

Keywords: cell culture medium; iron; low impurity; raw material impurity; recombinant protein product quality.

FIGURE 1

Effect of FAC iron source dose response in CCM on cell performance of cell line 1 in fed‐batch process. CHO K1 cells were cultivated in medium supplemented with either 2, 10, 50, or 100 mg Fe/L (FAC). (a) VCD in % normalized to the highest value. (b) Viability in %. (c) IgG concentration in % normalized to the highest value. (d) Iron concentration in mg/L. (e) Ammonium concentration in mmol/L. The horizontal dotted line in panel (e) represents the lower limit of detection. Data present mean ± SD of six biological replicates

FIGURE 2

CQAs of mAb1 produced by cell line 1 on day 10 of FAC dose response fed‐batch process. (a) HMW and main peak level in %. (b) N‐glycosylation forms (terminal sialylated, terminal galactosylated, terminal GlcNAc, and terminal mannosylated) in %. Data are mean ± SD of two replicate pools (each with three biological replicates)

FIGURE 3

Effect of ammonium dose response in CCM on cell performance of cell line 1 and on CQAs of mAb1. CHO K1 cells were cultivated in medium supplemented with 2 mg Fe/L (FAC) and supplemented with either 0 (=positive control), 0.236, 1.400, or 2.855 mM ammonium in the form of ammonium chloride. CQAs of mAb1 were determined on day 10 of fed‐batch process. (a) VCD in % normalized to the highest value. (b) Viability in %. (c) IgG concentration in % normalized to the highest value. (d) Ammonium concentration in mmol/L. (e) HMW and main peak level of mAb1 in %. (f) N‐glycosylation forms (terminal galactosylated and terminal GlcNAc) of mAb1 in %. The horizontal dotted line in panel (d) represents the lower limit of detection. Data present mean ± SD of four (a, b, c, and d) or two (e and f) biological replicates

FIGURE 4

Effect of different iron sources, FAC and FC, in CCM on cell performance of cell line 1 in fed‐batch process. CHO K1 cells were cultivated in medium supplemented with either 2, 10, 50, or 100 mg Fe/L (FAC or FC). (a) VCD in % normalized to the highest value. (b) Viability in %. (c) IgG concentration in % normalized to the highest value. Data present mean ± SD of six biological replicates

FIGURE 5

Effect of high and low manganese‐contaminated FC iron sources in CCM on cell performance of cell line 1 and on aggregation and glycosylation profile of mAb1. CHO K1 cells were cultivated in medium supplemented with either 10, 50, or 100 mg Fe/L (FC_Synt (low manganese impurity) or FC_Purch (high manganese impurity)). Additionally, for each tested iron concentration a third condition was prepared, where manganese was added to FC_Synt to achieve the same manganese concentration as present in FC_Purch. CQAs of mAb1 were determined on day 10 of fed‐batch process. (a) VCD in % normalized to the highest value. (b) Viability in %. (c) IgG concentration in % normalized to the highest value. (d) HMW and main peak level of mAb1 in %. (e) N‐glycosylation forms (terminal galactosylated and terminal GlcNAc) of mAb1 in %. Data present mean ± SD of six biological replicates (a, b, and c) or two replicate pools (each with three biological replicates) (d and e)

FIGURE 6

Effect of manganese dose response in CCM on cell performance of cell line 1 and on glycosylation profile of mAb1. CHO K1 cells were cultivated in medium supplemented with 10 mg Fe/L (FC_Synt (low manganese impurity)) and 0 (=positive control), 0.02, 0.25, 0.49, or 0.98 μM Mn²⁺ in the form of manganese (II) chloride. N‐glycosylation profile of mAb1 was determined on day 10 of fed‐batch process. (a) VCD in % normalized to the highest value. (b) Viability in %. (c) IgG concentration in % normalized to the highest value. (d) N‐glycosylation forms (terminal galactosylated and terminal GlcNAc) of mAb1 in %. Data are mean ± SD of six biological replicates (a, b, and c) or two replicate pools (each with three biological replicates) (d)

FIGURE 7

Effect of high and low manganese‐contaminated FC iron sources in CCM on cell performance of cell line 2 and 3 in fed‐batch process. CHO K1 cells (cell line 2) and CHOZN® cells (cell line 3) were cultivated in medium supplemented with either 2, 10, or 50 mg Fe/L (FC_Synt (low manganese impurity) or FC_Purch (high manganese impurity)). Additionally, for each tested iron concentration a third condition was prepared, for which manganese was added to FC_Synt to achieve the same manganese concentration as present in FC_Purch. Cell line 2: (a) VCD in % normalized to the highest value. (b) Viability in %. (c) IgG concentration in % normalized to the highest value. Cell line 3: (d) VCD in % normalized to the highest value. (e) Viability in %. (f) Fusion protein concentration in % normalized to the highest value. Data present mean ± SD of four biological replicates

FIGURE 8

Glycosylation profile of mAb2 and fusion protein produced by cell line 2 and cell line 3, respectively, on day 10 of fed‐batch process upon usage of high and low manganese‐contaminated FC iron sources (FC_Purch and FC_Synt) in CCM. (a) N‐glycosylation forms (terminal sialylated, terminal galactosylated, terminal GlcNAc, and terminal mannosylated) of mAb2 in %. (b) N‐glycosylation forms (terminal sialylated, terminal galactosylated, terminal GlcNAc, and terminal mannosylated) of fusion protein in %. Data are mean ± SD of two replicate pools (each with two biological replicates)