Development and Comparison of Alternative Methods to Purify Adalimumab Directly from the Harvested Cell Culture Fluid

Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.


INTRODUCTION
The United States Food and Drug Administration (US FDA) approved the first monoclonal antibody (mAb) in 1986. Since then, the market for therapeutic antibody drugs has experienced explosive growth as new drugs have been approved for treating various human diseases, including cancer, autoimmune, metabolic, and infectious diseases (1). The global therapeutic mAb market was valued at approximately US$ 115.2 billion in 2018 and is expected to generate revenue of US$ 300 billion by 2025 (2). The widespread application of mAbs in clinical practice requires the development of cost-effective production routes. In general, a significant percentage (50-80 %) of the total manufacturing cost of mAb is incurred during the downstream processing. Critical challenges in producing new antibody therapeutics include improving process economics and efficiency, furthermore, fulfilling the increasingly demanding quality criteria for FDA approval (3). Although a wide range of technology is available for the downstream processing of proteins, most mAb purification systems are based on the use of Protein A affinity chromatography (3). Protein A affinity chromatography is highly selective for antibodies, allowing the removal of more than 95 % of the impurities from a complex fermentation broth culture in one step (4). However, using Protein A has some disadvantages, which are essentially related to the high cost and sensitivity of Protein A resins and possible leakage of the ligand from the resin matrix (5). In most industrial processes, two further chromatography steps, typically a cation and an anion exchange step, are inserted to remove remaining host cell proteins, DNA, leached Protein A ligands, and mAb aggregates from the eluate of the Protein A chromatography step (6). Furthermore, the process also includes virus inactivation steps Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
3 by using low pH and virus filtration steps to ensure an acceptable level of virus clearance (4,7,8).
Since Protein A chromatography is considered the most critical and costly step in the general purification process of mAbs, intensive research efforts have been made in the past years to develop alternatives, including selective extraction or precipitation methods. Partitioning in aqueous two-phase systems (ATPSs) has proved to be a valuable tool for separating and purifying mixtures of biomolecules by extraction. ATPS is an ideal technology where protein clarification, concentration, and partial purification can be integrated into one step. This purification technique can be highly selective and can be suited for continuous operation on a large scale, thus allowing wider biotechnological applications. Both polymer-polymer (e.g., polyethylene glycol (PEG)-dextran, PEGstarch) and polymer-salt (e.g., PEG-phosphate, PEG-citrate) ATPSs have been developed and studied to purify different immunoglobulin proteins (9). The ATPS is a good solution to provide a moderate environment for antibodies, as both phases (polymer and salt) have high water content (80-90 %). In addition, phase-forming polymers can stabilize the tertiary structure of the target protein (10). Sequential precipitation of impurities and mAb in a selective manner is another promising strategy to substitute Protein A chromatographic separation. The precipitation method offers a fast process for protein separation with high yield. It is easy to scale up and operates with low costs (11).
However, to achieve sufficient purity of the recovered mAb fraction, a combination of different precipitating agents (e.g., CaCl2 and PEG) in sequential precipitation steps would be required. The addition of CaCl2 to precipitate high molecular weight impurities (HMWI) such as dsDNA, host cell proteins (HCPs), and protein aggregates prior to the selective precipitation of mAbs by using PEG as a precipitating agent was found to be an interesting strategy for efficient, cost-effective purification of mAbs (12). Precipitation of mAbs by addition of PEG can be combined with other precipitating agents, such as caprylic (octanoic) acid, to enhance the final purity of the recovered antibody (13).
Although promising results were achieved in the field of mAb purification by using extraction and precipitation methods, the yield and purity of mAbs should be significantly enhanced for industrial implementation. Optimization of the different process variables affecting the purification of mAbs by extraction and precipitation is a prerequisite to make these methods competitive with the Protein A chromatographic step. The purification of mAbs by using ATPSs is strongly affected by the characteristics and concentration of the phase-forming agents, ionic strength, and pH, among other factors (13,14). The efficiency and selectivity of PEG precipitation of mAbs are highly dependent on the molecular mass and concentration of the PEG molecule, and the pH applied. Several studies have been dedicated recently to investigate the effects of different factors on the performance of mAb purification by extraction (15) and precipitation methods (13,16), and optimizing these strategies Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
4 (10,14,17). However, major part of the available studies focused on the investigation of model systems, and in many cases, the target immunoglobulin proteins were not specified (10,14,17). Thus, limited data are available in the literature regarding the purification of certain monoclonal antibodies from real fermentation broth (16). In addition, most of the studies focused on investigating and developing one alternative recovery method of the selected protein, making the exact comparison of different purification methods extremely hard and uncertain.
Thus, the aim of this work was to investigate the effects of different process variables on the recovery and purification of adalimumab from cell-free fermentation broth by using chromatographic, extractive, and precipitation-dissolution methods. Adalimumab recovery by alternative purification methods was optimized, and these methods (extraction and precipitation-dissolution) were compared to each other and with the conventional chromatographic method. The comparison was based on the adalimumab yield obtained during purifications, the amount of remaining dsDNA and HCP, and the presence of aggregates.

Harvested Cell Culture Fluid
Fermentation broth containing adalimumab was derived from fed-batch cultures of recombinant Chinese hamster ovary (CHO) cells. The fermentation process was performed following the method detailed by Júlia Domján et al. (18). The fermentation broth was centrifuged at 4000 rpm for 40 minutes (Rotanta 460R, Hettich, Germany) at 20 °C and filtered through a bottle top vacuum filtration PES membrane (VWR International, Hungary) having a pore size of 0.2 µm to remove cells, cell debris and other insoluble contaminants. The obtained supernatant was stored in a refrigerator (-20 °C) until further use.

Purification methods
Protein A affinity chromatography HiTrap® MabSelect SuReLX resin (4.24 mL, GE Healthcare, Chicago, US) was filled into a glass column (10 cm × 5.5 cm) for the preparative purification of adalimumab from the fermentation broth by using Äkta pure fast protein liquid chromatography (FPLC) system (GE Healthcare, Uppsala, Sweden) at room temperature (25 °C). The column was equilibrated with 4 column volumes (CVs) of equilibration buffer (20 mM sodium phosphate,15 mM sodium chloride, pH=7.4). at a flow rate of 1 mL/min and the sample (120 mL) was loaded at 1 mL/min. Thereafter, the column was washed with 6 CVs of washing buffer (10 mM EDTA, 1.5 M sodium chloride, 40 mM sodium phosphate, pH=7.4) Food Technology and Biotechnology 61 (3) 2023 www.ftb.com.hr Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing. 5 at 1 mL/min flow rate. Elution of adalimumab was performed with 10 CVs elution buffer (100 mM sodium citrate, pH=3.3) at 1 mL/min flow rate. The eluate was collected, and its pH was adjusted to 5.00 with 1 M NaOH solution. Until further analysis, the eluate was stored in a refrigerator (-20 °C).
Quality parameters of the column, such as the asymmetry factor and the height equivalent to the theoretical plate (HETP), were examined before adalimumab purifications by following the protocol of the producer (19). Measurements were performed in down-flow modes at three different flow rates (1.5 mL/min, 2.0 mL/min, and 2.5 mL/min). Measurement of dynamic binding capacity (DBC) was performed using purified adalimumab solution by following the method detailed by the producer of the resin (20), and the influence of residence times (3.6 min, 2.05 min, and 1.44 min) on DBC was analyzed. The loading of purified adalimumab solution was stopped when the concentration of mAb in the column effluent (detected at UV 280 nm) was 10 % relative to the mAb concentration of the feed. This loading is defined as 10 % breakthrough, and DBC was determined at this point.

Cation exchange chromatography
The Protein A eluate was loaded onto a 5.9 mL packed POROSTM 50 HS column (Thermo Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

6
PEG-buffer (PEG-phosphate and PEG-citrate) aqueous two-phase extractions were performed as a one-step, batch extraction process to recover adalimumab in the PEG phase from the cell-free fermentation broth. The PEG-buffer extraction systems were prepared by mixing the appropriate amount of PEG3350 stock solution, phosphate or citrate buffer stock solution, fermentation broth, NaCl, and water. PEG3350 stock solution contained 50 w/w % PEG having an average molar mass of 3350 Da (PEG3350). The concentration of the phosphate and citrate buffer stock solutions was 40 w/w %, and they were prepared by using an appropriate amount of dipotassium hydrogen phosphate and sodium dihydrogen phosphate, or citric acid and tri-sodium citrate, respectively, according to the required pH. Phosphate buffer stock solutions with pH=6 and 8 and citrate buffer stock solutions with pH=5 were used in this study. The pH of the system was assumed to be equal to the pH of the stock solution applied. The extraction system contained 7 w/w % PEG3350 and 25 w/w % fermentation broth in all the cases, while the amount of NaCl, phosphate, or citrate and the pH of the system was varied ( Table 1). The extraction systems were supplemented with an appropriate amount of water to reach the final total weight of 10 g in all cases. The extractions were performed in 15 mL centrifuge tubes in triplicates. After adding all the components of the extraction system, it was properly mixed by vortex, three times each for 20 seconds. The mixed system was kept at 25 °C for 5 minutes and then centrifuged (Rotanta 460R, Hettich, Germany) at 5000 g for 10 minutes to separate the PEG and buffer phases. The PEG and buffer phases were analyzed for adalimumab content. Table 1 Two-step, PEG-dextran, and PEG-buffer aqueous two-phase extractions The two-step extraction process consisted of a first step referred to as the extraction step and a second step referred to as the back-extraction step, performed in PEG-dextran and PEG-phosphate extraction systems, respectively. After the extraction step, the major part of the adalimumab was obtained in the PEG phase, so the PEG phase was separated and subjected to the back-extraction step (Fig. S1). Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing. 7 (Na2HPO4; Sigma-Aldrich; Germany) in the appropriate ratio. The extraction (first extractive step of the two-step extraction process) was conducted at room temperature (25 °C) with 10 g total weight in 15 mL centrifuge tubes. All the components of the system were thoroughly mixed in a vortex for 20 seconds three times. The mixed system was kept at 25 °C for 5 minutes and then centrifuged (Rotanta 460R, Hettich, Germany) at 5000 g for 10 minutes. The bottom (dextran-phase) and top (PEG-phase) phases were separated, and the volumes and weight of the phases were measured. The top phase was subjected to the back-extraction process step and/or analysed to determine the adalimumab concentration. Designed experiments were performed to optimize selected process variables of the extraction step, such as pH (5,8), PEG to dextran ratio (6 %:10 %; 8 %:5 %), and concentration of NaCl (0 %; 1 %).
Back extraction was performed in a PEG-buffer system with a total weight of 0.7 g in 2 mL Eppendorf tubes at room temperature (25 °C). The back-extraction system was composed of 0.5 g of PEG-phase derived from the extraction step and 0.2 g, 0.35 g, or 0.5 g of buffer phase (40 w/w % phosphate buffer with pH=8). The system was mixed in a vortex for 20 seconds three times and then centrifuged at 5000 g for 10 minutes. The bottom and top phases were separated, and their volumes and weights were determined. The bottom phase (buffer phase) was analyzed for adalimumab concentration. Extraction and back-extraction process steps were performed in triplicates.

Precipitation-dissolution method
The investigation and development of a precipitation and dissolution method for adalimumab purification from fermentation broth were based on the work of Sommer et al. (17). In this study, adalimumab recovery from cell-free CHO fermentation broth by a precipitation-dissolution method was developed. Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
8 incubated for 60 min at 5 rpm. The obtained mAb precipitate was separated by centrifugation at 4000 g for 15 min. The supernatant was discarded, and the precipitate was dissolved in 0.75 mL histidine buffer (20 mM Histidine; 100 mM NaCl) (Fig. S1). Liquid samples were analyzed to determine adalimumab, HCP, and dsDNA concentrations. Precipitation experiments were performed in triplicates.

Adalimumab quantification
Determination of monomer adalimumab and small aggregates was performed by analytical size-exclusion chromatography (SEC). It was performed using a TSK-GEL G3000SWXL column The samples were excited at 480 nm, and the fluorescence emission intensity was measured at 520 nm with a spectrofluorometer (Thermo Scientific, Waltham, USA).

RESULTS AND DISCUSSION
Food Technology and Biotechnology 61 (3) 2023 www.ftb.com.hr Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

9
Purification of adalimumab by chromatography Before affinity chromatography purification of the fermentation broths on the Protein A column, quality parameters of the column were examined to verify the suitability of the filled resin for the chromatographic separation and to determine the appropriate flow rates for adalimumab purification.
In general, an asymmetry factor between 0.8 and 1.8 is considered acceptable in this case (23). Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
10 0 mM to 50 mM with a gradient of 1.67 mM NaCl/CV, respectively (Fig. S2). The peak of the main fraction was eluted at around pH=8.3 (adalimumab pI=8.2) and the resolutions from the peaks of acidic and basic charge variants were 1.14 and 1.96, respectively. The adalimumab yield (in the main fraction of monomer adalimumab) of the cation exchange chromatography was 90 % and the amount of aggregates were 0.36 % of monomer adalimumab. Thus, affinity and cation exchange chromatography resulted in an overall adalimumab yield of 82 %.

Adalimumab purification by extraction and back-extraction
Extraction in PEG-buffer aqueous two-phase system In the first step of investigating PEG-buffer systems in a one-step extraction of adalimumab from the cell-free fermentation broth, buffers of three different pH values were tested: pH=5 citrate buffer; pH=6 and 8 phosphate buffers (Table 1, Exp. 1-3). The highest adalimumab yield in the PEG phase (60 %) was obtained with a pH=5 citrate buffer. However, the results showed high standard deviations, which might be caused by partial precipitation of adalimumab during these extractions.
Precipitation probably occurred because of the high ionic strength in the system, which could be caused by the high proportion of NaCl together with the high concentration of buffer components.
Thus, the experiment was performed with pH=5 citrate buffer containing a reduced amount of NaCl (12.5 and 10 w/w % instead of 15 w/w % (Table 1, Exp. 4 and 5)). However, precipitation was still observed in these cases and the adalimumab yield significantly decreased in the case of the 10 w/w % NaCl system (45 %) compared to the previous measurements. As the presence of NaCl in an appropriate amount is considered to be essential for the efficient extraction of adalimumab to the PEG phase (25), ionic strength was reduced in the next step by decreasing the concentration of buffer components (8 w/w %), while the amount of NaCl (5, 10, and 15 w/w %) was varied (Table 1, Exp. 6-8). However, the achieved adalimumab yields were even lower (< 35 %) than in the previous experiments, and precipitation of adalimumab was still observed. Precipitation and partition of proteins in a polymer-buffer ATPSs might be strongly influenced not only by the ionic strength of the given phase but the type of the present ions, among many other factors (26,27). In our system, none of the investigated settings provided suitable conditions for efficient extraction of adalimumab into the PEG phase without considerable precipitation. Based on these results, this PEG-buffer system seems to be inefficient for adalimumab purification from our fermentation broth. In contrast, other investigations by using PEG-buffer (phosphate and citrate buffer) systems for mAb purification Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

11
(from Excellgene, Switzerland), and undefined antibodies were purified by PEG-buffer systems. In conclusion, although PEG-buffer systems work well in the case of different mAb solutions, it was not suitable with our fermentation broth containing adalimumab.
Extraction in a two-step process containing PEG-dextran and PEG-buffer aqueous two-phase systems.
The use of a polymer-polymer system could be preferred over a polymer-buffer system due to the lower ionic strength and the protein stabilizing effect of the phase-forming polymers. Thus, the PEG-dextran system was selected to investigate the extraction of adalimumab from cell-free CHO fermentation broth. Sequential extraction and back-extraction steps were investigated during the purification process. The extraction step was performed in the PEG-dextran system, and the backextraction step was performed in the PEG-phosphate system. During the extraction step, the effects of pH (5,8), NaCl concentration (0 %; 1 %), and PEG-dextran weight ratio (6 %:10 %; 8 %:5 %) on the adalimumab yield in the PEG phase were studied. The experiments were performed according to 2 3 experimental design. Based on the statistical evaluation, all factors (pH, NaCl concentration, PEG:dextran weight ratio) and their interactions significantly affect adalimumab yield (Fig. S3). The presence of NaCl (1 %) significantly improved the adalimumab yield and the PEG: dextran ratio of 8 %:5 % resulted in higher adalimumab yields compared to the ratio of 6 %:10 %. Furthermore, extractions performed at pH=8 were more favorable compared to pH=5 in terms of adalimumab yield (data not shown). Maximum adalimumab yield (75 %) was achieved with 1 % NaCl, pH=8, and 8 %:5 % PEG: dextran ratio. In the next step of our investigation, the effect of increasing the NaCl concentration (1 %, 2.5 %, and 5 %) on the adalimumab yield in the PEG phase was examined during the extraction step. By increasing the NaCl concentration, the adalimumab yield continuously increased in the PEG phase and decreased in the dextran phase. The adalimumab yield in the PEG phase was increased from 75 % to 95 % by increasing the NaCl concentration from 1 % to 5 % (Fig.   1). Adalimumab was enriched in the PEG phase during the extraction step, then fresh phosphate buffer (pH=8) was added to the separated PEG phase during a back-extraction step. Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

12
of NaCl concentration, an opposite trend was experienced during the back-extraction compared to the extraction step: with increasing the NaCl concentration (in the extraction step), the adalimumab yield in the buffer phase decreased during the subsequent back-extraction step (Fig. 2). Fig. 2 Moreover, precipitation was observed in the case of 5 % NaCl. Regarding the weight ratios of the PEG and buffer phases applied, increasing adalimumab yield was observed by decreasing the amount of the buffer phase (Fig. 2). Increased amounts of the added buffer phase (1:0.7 and 1:1 PEG: buffer ratios) caused adalimumab precipitation during the back-extraction steps, which explains that the lowest amount of buffer phase (1:0.4 PEG: buffer ratio), where no precipitation was observed, resulted in higher adalimumab yield. The highest adalimumab yield (91 %) during back-extraction was achieved by using a PEG: buffer weight ratio of 1:0.4 and 1 % NaCl in the extraction step. Although the highest yield of adalimumab was obtained with 5 % NaCl concentration during the extraction step, the 1 % NaCl extraction system was the best, with 91 % yield at the back-extraction step. The overall adalimumab yield of the extraction process, including the extraction and back-extraction steps, was calculated for all the cases, and the highest overall adalimumab yield (68 %) was achieved by using 1 % NaCl in the extraction step and a PEG: buffer ratio of 1:0.4 in the back extraction step. Thus, a two-step extraction process containing sequential extraction steps of PEG-dextran and PEG-buffer ATPSs was developed in our study, and it was found as promising strategy for the recovery of adalimumab from the fermentation broth produced in our laboratory. Comparable results have been presented for one-step extractions of IgG by using ATPSs based on PEG and dextran. Azevedo et al.; (28) achieved a 95 % yield of IgG in the upper (PEG) phase with an ATPS containing 5 % dextran, 8 % PEG with triethylene glycol diglutaric acid, and 10 mM phosphate buffer (pH=7). Rosa et al. (29) achieved 96 % of IgG in the PEG-rich phase in a single-stage extraction step, where the system was composed of 7 w/w % PEG (PEG3350), 5 w/w % dextrans, and 1.3 w/w % triethylene glycol diglutaric acid. In contrast to the one-step purifications, extraction systems containing two sequential steps have been barely studied so far, and they mainly focused on PEG-buffer systems in both steps (10,30).  Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

Adalimumab purification by precipitations and dissolution
Recovery of adalimumab from cell-free CHO fermentation broth was also investigated by using the precipitation-dissolution method. During the optimization of the process parameters affecting the recovery and purity of adalimumab obtained after the sequential precipitations and dissolution, one factor was changed at a time. As shown in Fig. S1, the process contained two sequential precipitations and a dissolution step. The first precipitation step was accomplished by adding CaCl2 to remove high molecular weight contaminants (e.g., dsDNA and aggregates) (31,32). This was followed by the precipitation of adalimumab with PEG solution. The main goal of this step was to separate mAbs from lower molecular weight impurities (e.g., HCP) (17). The last step was the dissolution of the precipitated adalimumab in histidine solution.
The effect of different CaCl2 concentrations (1.25, 2.5, 3.75 mM) was investigated in the first step. Following the precipitation with CaCl2, the obtained supernatant was analyzed for adalimumab and dsDNA concentrations. The adalimumab content of the supernatant did not decrease significantly, with yield higher than 98 % in all of the cases. However, the dsDNA concentration, relative to the mAb concentration, significantly decreased, but no significant difference was found in the dsDNA concentrations (1382 and 1408 ppm) when using 1.25 mM and 2.5 mM CaCl2 solution.
Thus, this treatment was selected as the first step to remove large molecular mass impurities such as dsDNA from the fermentation broth during all the following experiments.
In the next step, PEG-mediated precipitation of adalimumab was studied by varying the molecular mass and concentration of PEG and pH of the system. The obtained precipitates were separated and dissolved in 20mM histidine solution. Adalimumab yield was calculated based on the adalimumab content before the PEG-mediated precipitation and after the dissolution in histidine solution. The concentration of remaining dsDNA after the dissolution step was also determined.
Evaluation of the different settings was based on the adalimumab yield and dsDNA concentration.
The effect of the molecular weight of PEG was examined by using PEGs with an average molecular mass of 3350, 4000, and 8000 Da, using the same concentration (14 %) in all cases. As shown in Fig. 3, the highest adalimumab yield (84.3 %) and lowest dsDNA concentration (1027 ppm) were achieved by using PEG4000. Based on these results, PEG4000 was found to be suitable for adalimumab purification by the precipitation-dissolution method, so it was used in all of the following experiments.  Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
14 The effect of PEG content was investigated at 10 %, 12 %, 14 %, 16 %, and 18 % PEG concentrations. As shown in Fig. 4  In the last step, the pH of the precipitation step was varied within the range of pH=5.5-9.1 (Fig.   5). Investigating the adalimumab yield in the function of the pH applied during the precipitation step, a trend with a maximum adalimumab yield (85 %) at pH=7.5 was observed. In the meantime, the remaining dsDNA concentration showed an opposite trend in the function of pH, having a minimum value (1027 ppm) at pH=7.5. Thus pH=7.5 was chosen as the most suitable pH for adalimumab precipitation using 14 % PEG4000.
In summary, the investigated process of adalimumab purification by selective precipitationdissolution method contained two sequential precipitation steps and a dissolution step. The application of 2.5 mM CaCl2 was chosen for the first precipitation step to remove a considerable part of dsDNA and probably other high molecular mass impurities. In the second step of selective adalimumab precipitation, the following parameters were selected as the most favorable condition in  Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
After investigation of the novel purification methods based on precipitation and extraction, the most favorable conditions in terms of adalimumab recovery were selected for each method, and the purifications were performed in a new batch of cell-free CHO supernatant. During these experiments, the different purification processes were compared and contrasted in terms of adalimumab yield and the amount of HCP, dsDNA, and adalimumab aggregates in the final solutions ( Table 2). Table 2 HCP, dsDNA, and adalimumab aggregates were also determined after chromatographic purifications and in the initial fermentation broth ( Table 2)  , the precipitationdissolution method was unsatisfactory as an individual purification step; however, it could be a good process for pre-purification prior to other purification steps. In the case of the extraction-based purification method, both steps of extraction and back-extraction were examined. Compared to the precipitation-dissolution method, the extraction step resulted in similar adalimumab yield, but higher amounts of HCP, dsDNA, and adalimumab aggregates in the PEG phase ( Table 2). The subsequent back extraction step decreased the concentration of HCP and dsDNA relative to the mAb concentration, and almost completely eliminated protein aggregates (<0.1 %). However, the overall adalimumab yield was significantly decreased to 54 %. Interestingly, the relative HCP content after extraction-back extraction was even higher than the relative HCP content of the initial fermentation broth, indicating the necessity of further development of this process. On the other hand, considering the amount of small aggregates, extraction provided better results than chromatographic separation. Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
the optimization experiments and the experiments performed for the comparisons. In the latter, the fermentation broth contained less adalimumab (0.9 g/L instead of 1.3 g/L), which might negatively affect the achievable adalimumab yields during precipitations and extractions (13).
In summary, the precipitation-dissolution method and one-step extraction provided adalimumab yield comparable with the purification by affinity and cation exchange chromatography but with lower purity. Based on adalimumab yield and relative HCP and dsDNS contents, precipitation was found to be more promising compared to the extractive method for adalimumab purification from cell-free fermentation broth.

CONCLUSIONS
In this study, two alternative methods based on extraction and precipitation were developed and investigated to purify adalimumab directly from harvested cell culture fluid. The adalimumabcontaining fermentation broths were produced in our laboratory using CHO cell cultivations. The precipitation method contained two sequential steps of precipitation using CaCl2 and PEG, followed by dissolution in histidine solution. The extractive process contained two sequential steps of extraction and back-extraction based on PEG-dextran and PEG-buffer ATPSs, respectively. Both purification methods were optimized in several steps, and the best parameters were selected in terms of adalimumab yield and purity in certain cases. Classic chromatographic purification containing affinity and cation exchange steps was also performed and enhanced. The three different strategies were compared in terms of (monomer) adalimumab yield and the relative concentration of impurities such as HCP, dsDNA, and adalimumab aggregates. Subsequent affinity and cation exchange chromatography were found to be an efficient purification method for adalimumab resulting in high yield and excellent purity. However, there are several disadvantages of chromatographic purifications. One of the main drawbacks is the high price of affinity resins. A further disadvantage is that it is difficult to operate them in continuous mode, and the scale-up could also be challenging. To overcome these obstacles, alternative purification methods of precipitations-dissolution and extraction-back-extraction offer promising alternatives, and thus they were studied and compared with each other and with the chromatographic separation. The precipitation-based process resulted in good adalimumab yield however impurities with high concentrations were still present in the final solution. The extraction-based purification resulted in low adalimumab yield with low purity.
The precipitation-based method was found to be more promising for direct purification of adalimumab from harvested cell culture fluid compared to the extraction-based method. However, Food Technology and Biotechnology 61 (3) 2023 www.ftb.com.hr Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

17
further improvements are still required to compete with the performance of the chromatographic separations. However, considering the advantages of the alternatives (such as continuous operation, simplicity, and scalability), a cost-effective operation might be achieved without the same performance (e.g., mAb yield) that chromatographic purifications have. Also, the performance of these alternative mAb purifications might be significantly improved by combining them, which will be the goal of further studies.

FUNDING
Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

24.
MabSelect SuRe LX protein A resin | Cytiva. Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.  Please note that this is an unedited version of the manuscript that has been accepted for publication. This version will undergo copyediting and typesetting before its final form for publication. We are providing this version as a service to our readers. The published version will differ from this one as a result of linguistic and technical corrections and layout editing.