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Open Access

Peer-reviewed

Research Article

RETRACTED: Induction of heat shock protein expression in SP2/0 transgenic cells and its effect on the production of monoclonal antibodies

Retraction

After this article [1] was published, concerns were raised regarding the statistical analyses and interpretation of the results. Specifically:

  • Figs 1A, 2, 3A, 5 and 7A show statistical significance markers for all conditions including for the control group, despite the control group acting as the comparator for the other treatment conditions.
  • The Statistical analysis subsection of the Materials and methods section and the description of the statistical tests in the figure legends lack sufficient detail for replication.
  • The article presents statements and conclusions that are not supported by the results, including:
    • The expression of HSPs was greater in paracetamol-treated cells than in heat shock-treated cells.
    • Cells treated with paracetamol at concentrations above 0.5 mM showed a significant decrease in cell viability after 24 hours.
    • Heat shock treatment decreased cell viability after 24 hours.

The corresponding author stated that they agreed with the above concerns for the statistical analyses. During post-publication correspondence, they provided multiple statistical re-analyses for all graphs in [1]. A member of the PLOS One Editorial Board reviewed the author’s responses and repeated statistical analyses, and stated that these did not resolve the concerns raised for the statistical methods and that the re-analyses raised additional concerns for the reliability of the results reported.

Following editorial assessment, the PLOS One Editors concluded that the article does not meet the journal’s third and fourth criteria for publication [2]. In light of the above concerns, which call into question the validity and reliability of the published results, the PLOS One Editors retract this article.

All authors agreed with the retraction and apologize for the issues with the published article.

3 Mar 2026: The PLOS One Editors (2026) Retraction: Induction of heat shock protein expression in SP2/0 transgenic cells and its effect on the production of monoclonal antibodies. PLOS ONE 21(3): e0343942. https://doi.org/10.1371/journal.pone.0343942 View retraction

Abstract

The objective of the current investigation was to evaluate the induction of heat shock proteins (HSPs) in SP2/0 transgenic cells and the effect of these proteins on the production of monoclonal antibodies (mAbs). The SP2/0 cell line expressing the PSG-026 antibody, a biosimilar candidate of golimumab, the culture parameters, and the target protein expression were not justified for industrial production and were used for the experiments. Paracetamol and heat shock were used as chemical and physical inducers of HSPs, respectively. The results showed that paracetamol and heat shock increased the expression of HSP70 and HSP27 at the mRNA and protein levels. The expression of HSPs was greater in paracetamol-treated cells than in heat shock-treated cells. Paracetamol treatment at concentrations above 0.5 mM significantly reduced cell viability and mAb expression. However, treatment with 0.25 mM paracetamol results in delayed cell death and increased mAb production. Heat shock treatment at 45°C for 30 minutes after enhanced mAb expression was applied after pre-treatment with paracetamol. In bioreactor cultures, pretreatment of cells with paracetamol improved cell viability and shortened the lag phase, resulting in increased cell density. The production of mAbs in paracetamol-treated cultures was markedly greater than that in the control. Analysis of protein quality and charge variants revealed no significant differences between paracetamol-treated and control cultures, indicating that the induction of HSPs did not affect protein aggregation or charge variants. These findings suggest that inducing and manipulating HSP expression can be a valuable strategy for improving recombinant protein production in biopharmaceutical processes.

Citation: Jaffaraghaei M, Ghafouri H, Vaziri B, Taheri M, Talebkhan Y, Heravi M, et al. (2024) RETRACTED: Induction of heat shock protein expression in SP2/0 transgenic cells and its effect on the production of monoclonal antibodies. PLoS ONE 19(5): e0300702. https://doi.org/10.1371/journal.pone.0300702

Editor: Vara Prasad Saka, Dr. Anjali Chatterji Regional Research Institute for Homeopathy, INDIA

Received: December 12, 2023; Accepted: March 4, 2024; Published: May 2, 2024

Copyright: © 2024 Jaffaraghaei et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting information files.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

CHO, NS0, SP2/0, and PER.C6 cells are the most effective host cells for producing monoclonal antibodies [1]. While the maximum protein expression level in SP2/0 cells is approximately 5 times lower than that in CHO cells, the specific glycosylation profile of these cells can directly impact antibody activity. As a result, SP2/0 cells are considered a viable option for specific monoclonal antibodies [2]. Because glycosylation as a cQA can affect the immunogenicity, half-life, and pharmacokinetics of monoclonal antibodies (mAbs) [3], biosimilar producers usually use the same host cell as the original brand producer. Currently, SP2/0 cells are used as host cells for the production of mAbs such as Abciximab, Basiliximab, Canakinumab, Cetuximab, and Infliximab [1], as well as Golimumab and Ustekinumab [4]. Under stressful conditions, cells can protect themselves by inducing heat shock protein (HSP) expression, thereby increasing their chances of survival [5]. In bioreactors, cellular stress can lead to programmed cell death, as observed in various studies.

Cells can experience various types of stress when cultured in a bioreactor [6–8].

  1. Physical stress: Mechanical forces such as shear stress from agitation or turbulence within a bioreactor can impact cells. Physical stress can also result from the attachment of cells to surfaces or microcarriers.
  2. Chemical stress: Exposure to different chemicals in the culture medium, including changes in pH, osmolarity, toxins or metabolites, can stress cells.
  3. Oxygen and nutrient stress: Fluctuations in oxygen levels and nutrient availability within the bioreactor can challenge cell metabolism and function.
  4. Temperature stress: Variations in temperature within a bioreactor can affect cell growth and metabolism. Sudden temperature changes or high temperatures can be detrimental to cells.
  5. Oxidative stress: Reactive oxygen species (ROS) can accumulate due to oxygen exposure, leading to oxidative stress and potential cell damage.
  6. Shear stress: Cells can experience shear forces due to the movement of fluids in the bioreactor, which can affect cell viability and integrity.
  7. Hydrodynamic stress: Changes in fluid dynamics, including turbulence and flow rates, can stress cells and influence their behavior.
  8. Density stress: A high cell density in a bioreactor can lead to nutrient and oxygen depletion, affecting cell growth and viability.
  9. Biological stress: Interactions with other cells, such as competition for resources or the presence of pathogens or contaminants, can also stress cells.
  10. Environmental stress: Factors such as light exposure (in photobioreactors), vibrations, and noise within the bioreactor environment can potentially impact cell cultures [6–8].

Cells exhibit various responses to stress, ranging from activating cell survival pathways to programmed cell death. To resist environmental stress, cells in growth media employ different mechanisms, including the use of HSPs as natural protectors against environmental and physiological stressors [9]. Under stressful conditions, cells can protect themselves by inducing HSP expression, thereby increasing their chances of survival [10], but their upregulation can be damaging under pathological conditions [11, 12]. This adaptive ability has been conserved throughout evolution.

HSPs are responsible for preventing the formation of non-functional proteins, facilitating protein folding, protecting proteins from stress factors like temperature, pH, and low oxygen levels [13]; and preventing apoptosis. The production of cellular misfolded proteins is minimized by decreasing protein transcription and translation during the initiation of the heat shock response [14].

HSPs are vital for cellular protection and stress response and are categorized into classes such as HSP100 (protein disaggregation), HSP90 (chaperoning diverse proteins), and HSP70 (protein folding and transport). Additionally, HSP60/chaperonin assists in protein folding, and small heat shock proteins (HSPs) prevent aggregation. HSP110 aids in disaggregation, while functionally related proteins, such as CDC37, support protein stability and activation, contributing to the cellular stress response and quality control. Sustaining cellular homeostasis during stress requires these conserved proteins [15].

Numerous studies have been carried out to explore the expression of HSPs and their ability to enhance resistance in cells under stressful conditions [16, 17]. In a study aimed investigating the expression of HSP70 in normal and transfected NS0 cells, subjected to heat treatment for 15 minutes followed by an 18-hour recovery period, HSP70 expression was assessed. These findings indicated that expressing HSP70 resulted in a twofold increase in the survival of hybridoma clones. This improvement can be attributed to enhanced cell viability and a reduction in antibody degradation caused by proteases in the culture medium [18]. Comprehensive research on fed-batch techniques in CHO cells has shown that the use of micronutrients can induce cell apoptosis, which is detrimental to recombinant protein production at high cell densities [19]. Some studies have indicated that overexpressing HSPs, such as HSP27, HSP40 and HSP70, can increase protein expression and cell viability [20–22]. In one study, CHO cells engineered to overexpress HSPs showed increased interferon-gamma production, although cell viability decreased with longer culture durations [23]. Another study found that HSP27 overexpression in CHO cells that produce monoclonal antibodies led to increased cell density and antibody production without affecting glycosylation. HSP27 was found to interact with various cellular components and delay the activation of caspases, which are critical enzymes in apoptosis [24]. In another similar study, upregulating HSP70 in BHK-21 cells prevented apoptosis induced by nutrient deprivation or cytotoxic stimuli. In summary, overexpressing HSPs is crucial for preventing apoptosis, promoting cell survival, and enhancing protein production in CHO cells [25].

In addition to introducing additional copies of the HSP gene to cells, chemical induction can also be used to increase the expression of HSPs. Researchers have investigated the effects of various chemicals on HSP expression in different cell types [26]. In one study, L929 mouse cells were treated with chemicals such as sodium arsenite, cadmium chloride, and sodium salicylate at various concentrations and for various durations. The results showed that these chemicals boosted the replication of the HSP70 gene and increased mRNA translation [27]. Another study examined the impact of sodium salicylate on the accumulation of HSP105 and HSP70 in mammalian cells and revealed that the heat shock promoter was activated by treatment with sodium salicylate, which led to an increase in HSP expression. The prolonged use of therapeutic doses of sodium salicylate could stimulate HSP expression in mammalian cells, making it a promising therapeutic agent [28]. Additionally, a study in rat glioma cells evaluated seven different agents, including isopropanol, 1,4-dinitrophenol (DNP), diethylstilbestrol (DES), carbonylcyanide-m-chlorophenylhydrazone (CCCP), rotenone, paracetamol, and acetylsalicylic acid (ASA), as HSP inducers. After one hour of incubation at defined concentrations, HSP generation was induced by these agents. Cytotoxicity and the ability of these agents to induce HSPs were linked to their lipophilicity [29]. In summary, various chemical inducers that increase HSP expression, which can potentially be applied in response to cellular stress and therapeutic strategies, have been explored [30, 31].

Paracetamol was chosen to be the HSP inducer for transgenic SP2/0 cells producing PSG-026 in the present study. Paracetamol, known as acetaminophen, is commonly used to relieve pain and reduce inflammation and fever. Although the recommended maximum intravenous (IV) dosage of paracetamol for individuals weighing more than 50 kg is 4 grams/day (HIGHLIGHTS OF PRESCRIBING INFORMATION, FDA 2015), the amount of paracetamol used in the cell culture medium is low (< 0.3 mg/ml), and purification can significantly reduce the residual amount of paracetamol to lower levels. Various parameters, including cell culture conditions, PSG-026 expression, and HSP expression at the mRNA and protein levels, were evaluated. The quality profile of the final product resulting from the HSP induction treatment was compared with that of the nontreated samples.

Results

Effect of paracetamol concentration on HSP induction and protein expression in SP2/0 cells

The effect of paracetamol (ranging from 0 to 2 mM) as an HSP inducer was investigated at passages 2 and 3 in fed-batch-cultured SP2/0 cells. The growth curve and PSG-026 protein expression were evaluated as the primary screening responses. The maximum culture duration for the 0.25 and 0.5 mM paracetamol treatments was 6 days. However, for the 1.0 and 2.0 mM Paracetamol-treated cells, the cell viability sharply decreased during the first subculture (Figs 1, 2 and S1 Fig). Despite the decreased density of cells treated with 0.25 and 0.5 mM paracetamol, cell survival was delayed by one more day compared with that of the control untreated cells (Fig 1A, P values < 0.05; n = 3). The protein expression levels measured by protein A chromatography of the harvested supernatant indicated that the maximum monoclonal antibody (mAb) titer (372 μg/ml) was achieved in cells treated with 0.25 mM paracetamol. SDS–PAGE analysis of the diluted supernatant confirmed that the heavy and light chains of the expressed antibodies were comparable to the reference standard (S1 and S2 Figs). After initial screening of the paracetamol concentration and its cellular impact and protein expression, the 0.25 mM paracetamol treatment strategy was chosen for two subsequent subcultures.

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Fig 1. Cell viability and cell density curves.

(A) Effect of the concentration of paracetamol (0–2 mM), an HSP inducer, on fed-batch-cultured SP2/0 cells. The blue line is the control, the green line is 0.25 mM paracetamol, the orange line is 0.5 mM paracetamol, the pink line is 1 mM paracetamol, and the black line is 2 mM paracetamol. (B) Effect of the paracetamol concentration (0–2 mM), an HSP inducer on fed-batch cultured SP2/0 cells. The discontinuous blue line represents the control, the green line represents 0.25 mM paracetamol, the orange line represents 0.5 mM paracetamol, the pink line represents 1 mM paracetamol, and the purple line represents 2 mM paracetamol. The values are given as the means ± SDs (n = 3). P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, and ***p < 0.001 indicate a significant difference in cell viability according to a two-tailed t test.

https://doi.org/10.1371/journal.pone.0300702.g001

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Fig 2. Effects of paracetamol concentration (0–1 mM), an HSP inducer, on protein expression in passages 2 and 3 in fed-batch-cultured SP2/0 cells.

Protein A chromatography showed that the highest mAb concentration (372 μg/ml) occurred in cells treated with 0.25 mM paracetamol. SDS–PAGE analysis confirmed that the heavy and light antibody chains were comparable to those of the reference standard. Consequently, the 0.25 mM paracetamol treatment strategy was chosen for two subsequent subcultures based on its initial screening results. The values are given as the means ± SDs (n = 3). P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001 represent a significant difference according to a two-tailed t test.

https://doi.org/10.1371/journal.pone.0300702.g002

The results indicated that paracetamol concentrations above 0.5 mM reduce cell viability by less than 10%. Moreover, at 0.5 mM, paracetamol significantly decreased cell viability and cell density compared with those of the 0.25 mM condition. Consequently, the 0.25 mM concentration of paracetamol was chosen as the baseline for subsequent experiments. To determine the optimal duration for heat shock treatment to induce stress and apoptosis, cells were incubated at high temperatures (40 and 45°C) for four different durations (5, 15, 30, and 45 min) after two passages. The culture flasks were then transferred back to the normal condition (37°C), and the cell count and viability were determined at 24 and 48 hours post-treatment, as displayed in Table 1.

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Table 1. Conditions for heat shock.

https://doi.org/10.1371/journal.pone.0300702.t001

The findings revealed that cells cannot endure a heat shock at 45°C for more than 15 minutes. To ensure the application of an effective heat shock treatment without causing severe damage to the cells, 45°C for 30 minutes of incubation was selected for subsequent experimentation. To further investigate the effect of paracetamol on SP2/0 cells following heat shock treatment, an investigation was conducted according to S1 Table. The cell culture source was the same for all the treatments, with a cell density of 0.55 × 106 cells/ml and a viability of 98%.

The cell culture supernatant was diluted three times for SDS–PAGE analysis of the mAb expression level, and the cell pellet was utilized to detect HSP expression at both the mRNA and protein levels.

The growth curve demonstrated that cell death was delayed by one day in the paracetamol-treated group compared with the control group (Fig 3A and 3B, P values < 0.05, n = 3). Furthermore, there was a one-day difference in the duration of cell culture under heat shock conditions between the T groups treated with and without paracetamol.

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Fig 3. Cell viability and cell density in cell culture after different treatments.

(A) The effects of heat shock, paracetamol, and a combination of heat shock and paracetamol on the cell density of the culture and (B) the percentage of cell viability. The growth curve analysis in these figures shows that the paracetamol-treated group had a one-day delay in cell death compared with the control group. In addition, under heat shock conditions, there was a distinct one-day disparity in cell culture duration between the groups treated with and without paracetamol. This finding suggested a potential protective effect of paracetamol under these conditions. The values are given as the means ± SDs (n = 3). P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, and ***p < 0.001 indicate a significant difference in cell viability according to a two-tailed t test.

https://doi.org/10.1371/journal.pone.0300702.g003

The mAb titers for each condition are presented in Table 2. Paracetamol-treated and heat shock conditions produced the highest and lowest mAb titers, 486 and 245 μg/ml, respectively. Although thermal shock caused decreased mAb expression in T1 cells (245 μg/ml) compared with that in T2 cells, the impact of thermal shock was less pronounced in T3 cells, which underwent both treatments (412 μg/ml).

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Table 2. The mAb titer was detected by protein A chromatography for the T1, T2, and T3 conditions.

https://doi.org/10.1371/journal.pone.0300702.t002

SDS–PAGE analysis of the cell culture supernatants revealed protein bands comparable to those of the reference golimumab protein. There were more low-molecular-weight impurities in heat shock-treated cells than control cells (S2 Fig).

Quantitative HSP protein expression level

The expression level of HSPs in each condition was assessed by Western blotting (Fig 4A). The ratio of HSPs to β-actin was calculated. The folds of control in expression for each band was determined and is presented in Fig 4B and 4C (P values < 0.05, n = 3). Heat shock increased the expression ratios of HSP27 and HSP70 to 2.11- and 5.62-fold, respectively. However, treatment with paracetamol increased the ratio to 1.69- and 1.88-fold, respectively. Interestingly, after heat shock followed by pretreatment with paracetamol, the expression ratios of HSP27 and HSP70 were 3.15- and 2.70-fold, respectively. S5–S7 Figs show the original photos of the Western blot gels.

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Fig 4. Western blot analysis and comparison of HSP expression levels under different treatment conditions.

(A) Western blot analysis using specific antibodies against Heat shock, Para (Paracetamol), and Para + Heat shock (Paracetamol + Heat shock). However, after treatment with paracetamol, there was a significant increase in the expression of HSP70. Subsequent exposure to heat shock stress further increased the expression ratio of these genes. Remarkably, under experimental conditions in which heat shock stress was followed by pretreatment with paracetamol, we observed a significant increase in the expression ratio of HSP27 to HSP70. This interesting result suggested a synergistic effect between paracetamol and heat shock, potentially indicating a cooperative mechanism that enhances the cellular stress response mediated by these heat shock proteins. These findings provide valuable insights into the complex interplay between paracetamol and heat shock and between the modulation of HSP27 and HSP70 expression and illuminate potential avenues for further research into therapeutic strategies aimed at enhancing cellular stress resistance. The values are given as the means ± SDs (n = 3). P values < 0.05 were considered significant; *p < 0.05, **p < 0.01, ***p < 0.001 vs. Contrrol. The original images of the Western blot gels are shown in S5–S7 Figs.

https://doi.org/10.1371/journal.pone.0300702.g004

Quantitative HSP mRNA expression level

The 2-ΔΔCt method was used to analyze real-time PCR results. The expression of the β-Actin housekeeping gene was used to normalize the relative expression levels. The mRNA levels of HSP27 and HSP70 were elevated in all the treated cell groups compared with those in the control group (Fig 5; P values < 0.05; n = 3).

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Fig 5. Comparison of HSP mRNA expression levels under different treatment conditions.

Real-time PCR analysis, performed using the 2-ΔΔCt method and β-actin normalization, revealed increased mRNA levels of HSP27 and HSP70 in all treated cell groups compared with those in the control group (P values < 0.05). Notably, the T2 group presented significantly greater mRNA levels than did the T1 group (P values < 0.05), suggesting a more pronounced response. However, the T3 group exhibited no significant change compared with the T2 group (P values < 0.05), indicating potential response saturation. These findings elucidate the impact of treatments on heat shock protein mRNA expression dynamics. The values are given as the means ± SDs (n = 3). P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001 represent a significant difference according to a two-tailed t test.

https://doi.org/10.1371/journal.pone.0300702.g005

Furthermore, the increase in the mRNA levels of these two HSP mRNAs was significantly greater in the T2 group than in the T1 group (P values < 0.05). Interestingly, the mRNA levels of HSP27 and 70 in the T3 subgroup were not significantly different from those in the T2 subgroup (P values < 0.05).

The two fed-batch processes were performed in a 7 L bioreactor inoculated with paracetamol-treated cells as the seed according to the protocol described in Fig 6.

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Fig 6. Implementation of the fed-batch process using T2 cells in a 7 L bioreactor.

https://doi.org/10.1371/journal.pone.0300702.g006

All the process parameters, including temperature, dissolved oxygen (DO), pH, agitation, aeration, and feeding strategy, were based on the standard operating procedure for the bioreactor culture of PSG-026 at PersisGen Co. The results of three previously performed PSG-026 technical batches and the newly designed fed batches are depicted in Fig 7A and 7B (P values < 0.05, n = 3). The average maximum cell densities in the T2 and control groups were 5.4 × 106 and 3.9 × 106 cells/ml, respectively. During the control treatment, the average expression level was 392 mg/L, but it increased to 691 mg/L in the T2 treatment group (S2 Table and S3 Fig).

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Fig 7. Cell viability and cell density bioreactor (BR) culture.

The viability (A) and density (B) of cells in bioreactor cultures treated with paracetamol compared with those in previously performed PSG-026 technical batches. A and B show the outcomes of three prior PSG-026 technical batches and newly designed fed-batch experiments. In the T2 group, the average maximum cell density reached 5.4 × 106 cells/ml, while that in the control group was 3.9 × 106 cells/ml. This finding suggested that the T2 group exhibited increased cell density, potentially indicating the effectiveness of the experimental conditions in enhancing cell growth. The values are given as the means ± SDs (n = 3). P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001 represent a significant difference in cell viability according to a two-tailed t test.

https://doi.org/10.1371/journal.pone.0300702.g007

The protein profile of the harvested supernatant exhibited protein bands with molecular weights comparable to those of the reference standard, as shown in S3 Fig.

All clarified supernatants were subjected to protein A chromatography, and the eluate was assessed for purity and charge heterogeneity. The results of size exclusion HPLC indicated no significant changes in the number of aggregated forms between paracetamol-treated and control samples. The percentage of monomer forms in the standard golimumab sample was 98.6%, while in the control and treated samples was 98.2- and 98.6%, respectively (S4 Fig). The ion exchange HPLC (IEX-HPLC) results demonstrated comparable amounts of acidic and basic variants in control batch 07 and the protein A eluate of paracetamol-treated cells. The area percentage of the main peak in the control batch 07 and the paracetamol-treated protein A eluate were 46% and 48%, respectively (S4 Fig).

Discussion

The expression of HSPs has been observed in the C6 rat glioma cell line treated with paracetamol at different concentrations (70 to 100 mM), particularly at 90 mM [29]. Furthermore, studies have shown that paracetamol at concentrations of 3.3, 1.65, and 0.82 mM resulted in approximately 50% DNA fragmentation in lymphocyte cells after 24 hours [32]. In the present study, recombinant SP2/0 cells treated with paracetamol at concentrations above 0.5 mM showed a significant decrease in cell viability after 24 hours, and this change was accompanied by a decrease in protein expression (from 139 μg/ml in untreated cells to 97 μg/ml in cells treated with 1 mM paracetamol). Although both treatments with 0.5 mM and 0.25 mM Paracetamol were able to delay cell death for one day, the amount of protein expression was significantly higher in the 0.25 mM treatment (372 mg/L) compared to the 0.5 mM treatment (108 mg/L). This difference in protein expression could be due to the creation of stress conditions in the cells caused by the higher concentration of paracetamol. The stress conditions may have led to the utilization of cellular energy resources towards maintaining the physiological conditions of the cells, rather than protein expression.

Despite the decrease in cell viability in the control condition on the fourth day, the cell density did not decrease on the same days. The mechanisms that lead to increased cell density but decreased viability in mammalian cell culture can be attributed to various factors, such as contamination by microorganisms [33], cell senescence, and programmed cell death induced by environmental stress [34]. Considering that microbial monitoring of the cell culture process from the preparation of the cell bank to the end of the culture was carried out according to the standard operating procedure in the laboratory and that each initial culture experiment was performed from the active cell bank, the possibility of contamination or cell senescence was ruled out. However, it seems that the cells were under environmental stress under control conditions and despite cell proliferation and an increased cell number, their survival was affected.

The purpose of choosing heat shock conditions was to impose severe stress on cells and induce apoptosis. During heat shock treatment screening experiments, the cells could not tolerate 45°C for 30 minutes but survived at 40°C. The same conditions have been reported for the lymphocytes in which apoptosis was not induced at temperatures less than 42°C in less than 90 minutes, while temperatures above 43°C for 30 minutes or longer resulted in apoptosis [35]. According to prior research, C6 rat glioma cells exhibit increased expression of HSPs upon exposure to heat shock at 44°C for 30 minutes after a 1 hour incubation [29]. Cells activate various signaling pathways to promote resistance under stress conditions; however, if stress persists beyond cell tolerance, the cell death pathway is initiated [36]. Another study aimed to screen the effects of chemical HSP inducers on U937 human lymphoma cells under hyperthermic conditions (44°C for 15 min) as a control treatment [37]. This study revealed that, compared with those in the control treatment, the relative expression of HSP70 in the treated group was more than 2-fold greater. Consistent with the findings of previous studies, heat shock treatment decreased cell viability after 24 hours. However, when heat shock was applied after pretreatment with paracetamol (T3 group), cell death was delayed for one day, and recombinant protein expression increased to 412 mg/L. However, with the addition of paracetamol alone, viability and cell density decreased, and in addition to a one-day delay in cell death, the expression level reached 486 mg/L. SDS–PAGE profile analysis showed no changes in protein degradation or amount.

Ishihara et al. performed a study in which the viable cell density of mouse fibroblasts gradually decreased after heat shock treatment at 45°C for 45 minutes. However, pretreatment with salicylic acid, an HSP inducer, increased cell viability, and HSP70 may play a crucial role in preventing apoptosis [28]. In the present study, the relative expression of the HSP70 and HSP27 mRNA and protein was increased by both heat shock and paracetamol treatment. Furthermore, when heat shock treatment was applied to the paracetamol-treated cell culture, HSP70 and HSP27 mRNA levels increased compared with those in the cells treated with heat shock or paracetamol alone. These findings highlight the specific effects of paracetamol and heat shock on HSP induction. According to a similar study in which shikonin was used as an HSP inducer in U937 cells, the ratio of HSP70 expression to β-actin increased 3-fold [37]. Curiously, the more than 20-fold increase in the relative expression of HSP70 mRNA in the paracetamol-treated group resulted in an approximately 3-fold increase in HSP protein expression compared to that in the control group. It was also observed that the higher expression of HSP mRNA in the paracetamol-treated group than in the heat treatment group was not related to the HSP expression level. Moreover, the HSP70 expression level was significantly greater than the HSP27 expression level at both the mRNA and protein levels under each condition.HSP70 functions as a potent antiapoptotic factor capable of obstructing both the extrinsic and intrinsic routes leading to programmed cell death [38]. Overexpression of HSP70 has been indicated to confer resistance against apoptosis and boost the expression of recombinant factor VIII and the viable cell density on day 7 was greater in the rBHK-HSP70 strain than in with the rBHK-host strain [25]. Additionally, in CHO cells expressing IFN-γ, the overexpression of HSP27 and HSP70 gradually decreased cell viability from 36 to 72 hours in a fed-batch culture. Extending the cultivation time resulted in a 2.5-fold improvement in IFN-γ production compared to that in the control cells [23]. Furthermore, Ishihara et al. demonstrated that HSPs can be induced in fibroblasts through treatment with 60 mM sodium salicylate, which enhances cellular resistance to thermal stress during heat shock treatment at 45°C for 45 minutes [28]. Various chemical factors (supplements, metabolites, pH, and oxygen), as well as physical factors (shear stress, bubble size, and temperature), significantly impact mammalian cell growth parameters and the lag phase in bioreactor culture [39]. According to these reports, increasing the expression of HSPs through gene copy amplification or induction can be a valuable strategy for enhancing resistance against apoptotic changes, thereby facilitating the production of recombinant glycoproteins.

In the current study, pretreating the seed culture with paracetamol in two subsequent passages before transferring it to the bioreactor improved cellular stability during the initial stages of bioreactor culture. The cell growth curve for the pretreated culture showed a lag phase duration of one day less than that of the control condition. Although the initial cell density in the bioreactor was the same for both the control and paracetamol-treated conditions, the average cell density on the second day of culture in the paracetamol-treated condition was twice as high as that in the control condition. On the third day, cell viability decreased in the control group, while on fourth day, cell viability decreased in the paracetamol-treated group. Moreover, in a developed fed-batch bioreactor culture process for the production of Epratuzumab from SP2/0 cells, a maximum cell density of approximately 16 × 106 cells/ml was achieved on the sixth day, despite the lag phase continuing for three days [8]. There are two kinds of structural defects in proteins: modification by a chaperone system or elimination by ubiquitin [40]. Increasing the expression of heat shock proteins, which act as chaperones in cells, in addition to improving the resistance of cells to apoptosis, can improve the ability of cells to solubilize and correct the structure of misfolded or complex proteins [41]. This ability can ultimately improve the expression of proteins with the correct structure in transgenic cells.

Risk analysis utilizing the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICHQ6B) has shown that the amount of aggregate and charge variants in monoclonal antibodies are CQAs [42]. The aggregation of proteins can significantly impact potency [43], immunogenicity [44, 45], protein stability, and safety [43, 46]. Thus, it is essential to ensure that an acceptable level of aggregated proteins guarantees the efficacy and safety of the product [47]. The formation of protein complexes may arise from factors such as high protein expression levels, interactions of unfolded proteins, or inadequate chaperones to facilitate proper protein folding [4].

In the current study, despite the twofold increase in expression observed in the paracetamol-treated culture, size exclusion chromatography (SE-HPLC) analysis demonstrated no significant changes in the number of aggregated proteins between the paracetamol-treated and control conditions. Several modifications, including deamidation, sialylation, glycation, oxidation, cysteine-related modifications, and unformed disulfide bonds, were identified in the acidic and basic forms of the mAbs. Charge variants play a critical role in efficacy (complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity), safety (immunogenicity), and stability, which make them essential quality considerations [48]. It should be noted that medium composition is another significant factor that contributes to the formation of charge variants [49]. The content of charge variants can be influenced by other bioreactor cell culture parameters, such as temperature, pH, culture duration, and cell density [50, 51]. Additionally, the mixing rate, as a stress factor in bioreactor culture, can promote the formation of mAb aggregates and charge variants [52]. In our experiment, despite the increased cell density and protein expression observed following paracetamol treatment, there were no qualitative changes related to the protein-charged variants, which could suggest that the induction of HSPs does not significantly affect the formation of protein variants.

Materials and methods

Reagents

Primary antibodies against beta-actin (SC-47778), HSP70 (SC-660488), and HSP27 (SC-13132), as well as secondary antibodies (m-IgGk BP-HRP; SC-516102), were purchased from Santa Cruz Biotechnology. The chemicals and necessary kits used were obtained from the sites of their appropriate representatives and included SDS–PAGE sample buffer (Bio-Rad, Berkeley, CA, USA), 4–12% Bis-Tris protein gels (Bio-RAD), Coomassie protein stain (Expedeon, Swavesey, UK), a ProPac SCX-10 analytical column (Thermo Fisher Scientific, US), a TSKgel G3000SWxL size exclusion column (Tosoh Bioscience GmbH, Germany), an RNeasy Mini Kit (Qiagen, Germany), and a Protease Inhibitor Cocktail-P2714 (Sigma–Aldrich).

Cell culture

The SP2/0 single clone cell line expressing the PSG-026 antibody was kindly received from the Persis Gen Par Biopharma Accelerator (Persis Gen Par Co., Tehran, Iran). Hybridoma-SFM culture medium (Thermo Fisher, USA) supplemented with 5 mM L-glutamine (Gibco) was used as the basal medium, and HyClone Cell Boost 7b was used as the feed supplement.

The cell culture process started by thawing the cell in a vial at 37°C and resuspending them in 20 ml of medium in a 125 ml shake flask. The flask was then incubated at 37°C, 5% CO2, and 120 rpm. For fed-batch experiments, after two passages, 100 ml of culture medium was inoculated with 0.35 × 106 cells/ml and incubated at 37°C, 5% CO2, and 120 rpm until the day of harvest (sixth to eighth day). Feeding was performed by adding 2% of the initial volume on the second day until the day of harvest. In all the fed-batch experiments, cells were harvested when the viability decreased to less than 30%. The culture medium was centrifuged at 365xg (20 min) to harvest the supernatant for future investigations.

Paracetamol treatment

The effective and nonlethal doses of paracetamol were determined in a fed-batch experiment with 0, 0.25, 0.5, 1, and 2 mM paracetamol at the final concentrations. For the experiment, 1.0–1.5 106 cells/ml were divided into five groups. The fed-batch process was initiated for each condition by adding 1.0–1.5 × 106 cells/ml 48 hours after the second passage. Cell count and viability were monitored daily, and the medium was collected when the viability decreased to 70%. The protein titer was measured using Protein A chromatography. After the effective dose of paracetamol was determined, the cells were treated with the selected dose after two consecutive passages before the fed-batch process was started. The fed-batch process was executed following the conditions mentioned earlier. After harvesting, the cells were collected by centrifugation (2000 rpm, 5 min) and stored at -70°C for gene expression and western blotting studies. Protein A chromatography was used to purify the supernatant containing the target antibody from the medium, and the protein A concentration was analyzed through SDS–PAGE and ion exchange HPLC.

Heat shock treatment

For heat shock treatment, 24 h after the passage, the cells were incubated at 45°C for 15, 30, or 45 minutes and then returned to 37°C under normal conditions. The cell count and viability were monitored for two consecutive passages after the treatment. A condition that is both effective and lethal was chosen for additional combination experiments with paracetamol. In combination experiments, the cells were first subjected to heat shock and treated various concentrations of paracetamol. After 24 hours, the cells were transferred to 45°C for the appropriate duration and then returned to normal conditions. The cells were given paracetamol after two consecutive passages before the fed-batch process was started.

SDS–PAGE analysis

The protein samples obtained from different stages of cultivation and purification were diluted in SDS–PAGE sample buffer (Bio-Rad, Berkeley, CA, USA) containing 1% (v/v) 2-mercaptoethanol and heated for 5 minutes at 90°C. Subsequently, the samples were resolved on pre-cast Gel® Precast 4–12% Bis-Tris Protein gels (Bio-Rad) and stained using InstantBlue™ Coomassie protein stain (Expedeon, Swavesey, UK).

Protein A affinity chromatography

Protein A affinity chromatography was performed using a HiTrap MabSelect SuRe prepacked column (GE) to assess the protein expression level. The column was equilibrated with equilibrium buffer (20 mM sodium phosphate; pH = 7.4, conductivity of 12 ± 2 mS/cm). After the medium was added to the column, the mixture was washed with 3 column volumes (CVs) of equilibrium buffer. Once the UV spectrum reached the baseline, elution buffer (100 mM trisodium citrate, pH = 3.0, conductivity of 40 ± 3 mS/cm) was applied to the column. Then, the eluate peak was collected and filtered (Millex syringe filter, pore size 0.45 μm) before being subjected to other tests, such as IEX-HPLC and SE-HPLC.

Charge heterogeneity

IEX-HPLC analyses of charge variant comparability (CVCs) were conducted using a strong cation exchange (SCX) ProPac SCX-10 analytical column (Thermo Fisher Scientific, US) following an in-house protocol. Briefly, samples (2 mg/ml, 200 μl) were treated with carboxypeptidase B (0.1 mg/ml). A 2 mg/ml protein sample (35 μl) was injected into the HPLC column at a flow rate of 0.7 ml/min.: Column temperature: 40°C, and detection wavelength: 280 nm. After running mobile phase A (20 mM MES buffer, pH = 6.4), a linear gradient of mobile phase B (20 mM MES buffer, 1 M NaCl, pH = 6.4) (5–95%) was used to separate the charge variants. The analysis was carried out by comparing the peak patterns of Simponi® and expressed mAb samples for acidic, main-form, and basic variants. The results are quantitatively expressed as percentages of the total peak area.

Purity and integrity

Comparative SE-HPLC was employed to analyze the presence of high-molecular-weight (HMW) aggregation and low-molecular-weight (LMW) degradation protein impurities in PSG-024. A TSKgel G3000SWxL size exclusion column (Tosoh Bioscience GmbH, Germany) was used for the SE-HPLC analysis. The analysis was performed according to previously established methodology [53, 54]. The mobile phase contained 300 mM NaCl in 100 mM sodium phosphate buffer (pH = 6.7). The autosampler was adjusted to 2-8ºC, 20 μL, 0.5 ml/min, and 280 nm for the temperature, injection volume, flow rate, and absorbance wavelength.

Comparative analysis of the SE-HPLC peaks and retention times was conducted for the nontreated, treated, and Simponi® samples. The content of impurities was quantitatively measured as a percentage of the total peak area. Additionally, SDS–PAGE was utilized to detect size-related heterogeneities, especially the correct mAb assembly and protein degradation, under both reducing and nonreducing conditions.

Western blot analysis

For protein extraction, 1 × 106 cells from each condition were resuspended in lysis buffer (25 mM Tris-HCl, pH = 7.5; 150 mM NaCl; 1% NP-40; and 1 mM EDTA, pH = 8.0). Fresh 1 mM PMSF, 1 mM Na3VO4, and 1X Protease Inhibitor Cocktail-P2714 (Sigma–Aldrich) were added to the lysis buffer. The samples were vortexed for 5 min, followed by centrifugation at 13,000 × g at 4°C. The supernatant was collected, and the protein concentration was estimated using the standard Bradford method. The samples were then separated via SDS–PAGE and transferred onto a nitrocellulose membrane. The membrane was subsequently probed with primary antibodies including anti-HSP70 (SC-66048), anti-HSP27 (SC-13132), and anti-beta actin (SC-47778), followed by a secondary antibody, mouse anti-rabbit IgG-HRP (SC-2357) (Santa Cruz Biotechnology). The desired proteins were visualized using advanced ECL reagents (RPN2134, Cytiva). The density of each band was measured using Gel Analyzer version 19.01.

Real-time PCR analysis

Total RNA was isolated using the Qiashredder (Qiagen, Germany) and purified using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. The RNA concentration was measured using a NanoDrop spectrophotometer. Subsequently, 1 μg of RNA was converted to cDNA using an iScript™ cDNA was used synthesis kit. For quantitative PCR, 50 ng of cDNA in each reaction. Table 3 provides the designed primer pairs and their corresponding gene accession numbers. The annealing temperature for each primer pair was determined through gradient PCR and analyzed via agarose electrophoresis. The qPCR procedure was as follows: initial amplification at 95°C (5 min), followed by 45 cycles of 10 sec at 95°C and 30 sec at the specific annealing temperature for each primer set. This was followed by melting curve analysis, which consisted of 15 sec at 95°C, 15 sec at 65°C, and a final cooling to 40°C. The quantification cycle (Cq) value for each gene was normalized to that of the reference β-actin gene (2ΔCqBeta Actin-ΔCqGOI).

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Table 3. Sequences of primer used for qPCR.

https://doi.org/10.1371/journal.pone.0300702.t003

Statistical analysis

GraphPad Prism 8 (GraphPad, La Jolla, CA, USA) was used for all the statistical analyses. The data are displayed as the mean ± standard deviation (S.D.). One-way analysis of variance (ANOVA) was used to determine significance. Differences for which the P value was less than 0.05 were considered to be significant. To compare groups, two-tailed t test were used.

Supporting information

S1 Fig. SDS–PAGE analysis of protein expression in fed-batch cultured SP2/0 cells.

Reduced 10% SDS–PAGE analysis of protein expression in fed-batch cultured SP2/0 cells treated with different concentrations of paracetamol (0–2 mM), an HSP inducer. 1, reference golimumab protein; 2, supernatant of untreated cells; 3–5, supernatant from cells treated with 0.25, 0.5, or 1 mM paracetamol; M, protein molecular weight marker.

https://doi.org/10.1371/journal.pone.0300702.s001

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S2 Fig. Comparative analysis of treated SP2/0 cells by SDS–PAGE.

Comparative SDS–PAGE analysis of the effects of paracetamol and heat shock treatment on SP2/0 cells; M, protein molecular weight marker; 1 & 6, culture supernatant from untreated cells; 2 & 7, culture supernatant from T2 cells; 3 & 8, culture supernatant from T1 cells; 4 & 9, culture supernatant from T3 cells; 5 & 10, standard golimumab protein.

https://doi.org/10.1371/journal.pone.0300702.s002

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S3 Fig. Analysis of the SDS–PAGE results for the bioreactor culture supernatants.

SDS–PAGE analysis of bioreactor culture supernatants; M, protein molecular weight marker; 1 & 2, PSG-026 control batches of 7 & 8 groups; 3 & 4, batches on paracetamol-treated cells; 5, reference golimumab molecule.

https://doi.org/10.1371/journal.pone.0300702.s003

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S4 Fig. Comparison of protein purity and charge heterogeneity in cultured cells.

(A & D), Reference golimumab proteins; (B & E), Protein A eluates of Batch 07; (C & F), Protein A eluates of Paracetamol-treated batch N2. The clarified supernatants were subjected to protein A chromatography to evaluate purity and charge heterogeneity. Size exclusion HPLC revealed no significant differences in the aggregated forms of the compounds between the paracetamol-treated and control samples. The monomer percentages were 98.2% for the control, 98.6% for the standard golimumab, and 98.6% for the treated samples. Both control batch#07 and the Paracetamol-treated protein A eluate showed comparable levels of acidic and basic variants, with the main peak area percentages at 46% and 48%, respectively, according to IEX-HPLC.

https://doi.org/10.1371/journal.pone.0300702.s004

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S5 Fig. The original photo of Western blot gel (HSP27).

Illustrative Western blot analysis of HSP27 in the cell homogenate in Heat shock, Para (Paracetamol), and Para+Heat shock (paracetamol+Heat shock), and Control conditions.

https://doi.org/10.1371/journal.pone.0300702.s005

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S6 Fig. The original photo of Western blot gel (HSP70).

Illustrative Western blot analysis of HSP70 in the cell homogenate in Heat shock, Para (Paracetamol), and Para+Heat shock (paracetamol+Heat shock), and Control conditions.

https://doi.org/10.1371/journal.pone.0300702.s006

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S7 Fig. The original photo of Western blot gel (β-actin).

Illustrative Western blot analysis of β-actin in the cell homogenate in Heat shock, Para (Paracetamol), and Para+Heat shock (paracetamol+Heat shock), and Control conditions.

https://doi.org/10.1371/journal.pone.0300702.s007

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S1 Table. The combined effect of paracetamol (Pa) and heat shock (HS) treatment on SP2/0 cells: Experimental approach and cell culture details.

The times of the three subcultures are shown for P1, P2 and P3.

https://doi.org/10.1371/journal.pone.0300702.s008

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S2 Table. The influence of paracetamol treatment on cell density and protein expression in bioreactor cultures.

https://doi.org/10.1371/journal.pone.0300702.s009

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S1 Graphical abstract.

https://doi.org/10.1371/journal.pone.0300702.s010

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S1 Raw images.

https://doi.org/10.1371/journal.pone.0300702.s011

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S1 File.

https://doi.org/10.1371/journal.pone.0300702.s012

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