Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture

Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2014 www.elsevier.com/locate/jbiosc

Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture Shohei Kishishita,1, 3, * Tomoko Nishikawa,2 Yasuharu Shinoda,2 Hiroaki Nagashima,2 Hiroshi Okamoto,2 Shinya Takuma,2 and Hideki Aoyagi3 Project Planning and Coordination Department, Project and Lifecycle Management Unit, Chugai Pharmaceutical Co., Ltd., 1-1 Nihonbashi-Muromachi 2-Chome, Chuo-ku, Tokyo 1038324, Japan,1 API Process Development Department, Pharmaceutical Technology Division, Chugai Pharmaceutical Co., Ltd., 5-1 Ukima 5-Chome, Kita-ku, Tokyo 115-8543, Japan,2 and Life Science and Bioengineering, Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1 Tennodai 1-Chome, Tsukuba, Ibaraki 305-8572, Japan3 Received 21 August 2014; accepted 30 October 2014 Available online xxx

During the production of therapeutic monoclonal antibodies (mAbs), not only enhancement of mAb productivity but also control of quality attributes is critical. Charge variants, which are among the most important quality attributes, can substantially affect the in vitro and in vivo properties of mAbs. During process development for the production of mAbs in a Chinese hamster ovary cell line, we have observed that an improvement in mAb titer is accompanied by an increase in the content of acidic charge variants. Here, to help maintain comparability among mAbs, we aimed to identify the process parameters that controlled the content of acidic charge variants. First, we used a PlacketteBurman design to identify the effect of selected process parameters on the acidic charge variant content. Eight process parameters were selected by using a failure modes and effects analysis. Among these, temperature shift was identified from the PlacketteBurman design as the factor most influencing the acidic charge variant content. We then investigated in more detail the effects of shift temperature and temperature shift timing on this content. The content decreased with a shift to a lower temperature and with earlier timing of this temperature shift. Our observations suggest that PlacketteBurman designs are advantageous for preliminary screening of bioprocess parameters. We report here for the first time that temperature downshift is beneficial for effective control of the acidic peak variant content. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Acidic charge variants; Chinese hamster ovary cells; IgG monoclonal antibody; PlacketteBurman design; Temperature shift]

Chinese hamster ovary (CHO) cells are popular mammalian hosts for commercial production of therapeutic proteins (1). Among these therapeutic molecules, therapeutic monoclonal antibodies (mAbs) are the fastest growing items of interest. They are now used widely because their high antigenic specificity and low incidence of unfavorable side effects make them highly effective. Although mAbs have proven to be useful therapeutic products, the typical doses of these products required for treatment are markedly higher than those of most other biologics, resulting in the need for large-scale production and efficient, cost-effective manufacturing processes to reduce the cost to patients. In the past few years, mAb productivity has been enhanced by improving and refining clone selection, expression vectors, transfection technologies, and culture media. In the typical optimization of fed-batch processes, mAb expression levels of 1e5 g/L are currently achieved (2e4). Maintaining not only mAb productivity but also mAb quality is critical, because the mAb molecules produced from CHO cells contain heterogenous variants that have implications for efficacy

* Corresponding author at: Project Planning and Coordination Department, Project and Lifecycle Management Unit, Chugai Pharmaceutical Co., Ltd., 1-1 Nihonbashi-Muromachi 2-Chome, Chuo-ku, Tokyo 103-8324, Japan. Tel.: þ81 33 516 5570; fax: þ81 33 281 0217. E-mail address: [email protected] (S. Kishishita).

and the risk of adverse immune responses in patients. Heterogeneity in mAbs is represented by aggregation and the presence of charge variants, typically as a result of various post-translational modifications such as oxidation, deamidation, glycosylation, glycation, isomerization, succinimide formation, N-terminal pyroglutamic acid formation, or C-terminal lysine clipping (5e7). Among the various mAb features that result in heterogeneity, the content of charge variants is one of the most important, because charge variants can substantially affect the in vitro and in vivo properties of mAbs. For example, experiments using chemically modified mAbs have revealed that charge variants can alter binding to proteins or cell membrane targets, thus affecting the tissue penetration, tissue distribution, and pharmacokinetics of the mAbs (8e15). Therefore, mAb charge variant levels must be controlled precisely, but there is currently little information available on the control of these variants by using process parameters; moreover, to our knowledge there have been no reports on the control of acidic variant levels. During process development, we have observed that an improvement in mAb titer was accompanied by an increase in the content of acidic charge variants. Here, with the aim of maintaining comparability among mAbs, we investigated the process parameters affecting the acidic charge variant content of mAbs expressed in CHO cells. Our strategy was to first adopt a statistical experiment (PlacketteBurman design) for initial screening of process

1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.10.028

Please cite this article in press as: Kishishita, S., et al., Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.10.028

2

KISHISHITA ET AL.

J. BIOSCI. BIOENG.,

parameters affecting the acidic charge variant content. We then tested in detail the effect of the process parameter selected from the screening. MATERIALS AND METHODS Cell line and cell culture The cell lines used were generated from dihydrofolate reductase (DHFR)-deficient CHO cells (DXB11, a gift of Dr. L. Chasin) that had been adapted to growth in CHO-S-SFM II medium (Life Technologies, Grand Island, NY, USA). The expression system used was based on DHFR amplification (16,17). The cell lines were transfected with plasmids encoding different IgG mAbs. Cells were passaged regularly in mixed CHO-S-SFM IIeCD-CHO medium (50:50) (Life Technologies, Grand Island, NY, USA) supplemented with methotrexate. Cells were maintained in suspension in 125-mL Erlenmeyer shake flasks (Corning, NY, USA) on an orbital shaker (Yamato, Tokyo, Japan) rotating at 100 rpm; the shaker was placed in an incubator at 5% CO2 in air humidified at 37 C. Unless otherwise specified, the working volume in the 125-mL Erlenmeyer shake flasks was 50 mL. Production culture was performed in fed-batch mode with continuous feeding; model BCC or BCP bioreactors (125 mL or 1 L; ABLE, Tokyo, Japan) were used for production culture. Cells were inoculated at 2.0  105 cells/mL into the bioreactors. The dissolved oxygen concentration was controlled at 40% of air saturation, and the pH was controlled at 7.2 until the end of day 2 and at 6.9 thereafter by using mixed 1 M NaOHe1 M NaHCO3 solution and CO2. Continuous feeding was started on day 3. After production culture, the cell culture fluids were centrifuged (190 g, 5 min) and filtered (0.22 mm; polyethersulfone membrane) to remove the cells, and antibodies for the analysis of acidic charge variants were purified from the fluids by using a POROS A20 Protein A column (Life Technologies, Carlsbad, CA, USA). Basal and feed media CHO-S-SFM II e CD-CHO medium was used for passage and production culture. Highly concentrated CHO-S-SFM II e CD-CHO medium was used as a feed medium. Analytical methods Cell counting and cell viability determination were performed with a CEDEX model AS20 automated cell counting device (Roche Innovatis, Bielefeld, Germany), and trypan blue exclusion. mAb titers were measured by protein-A high performance liquid chromatography (Waters, Milford, MA, USA) assay with appropriate reference standards. We used a POROS A20 Protein A column (Life Technologies). The acidic charge variant content was determined by highperformance ion-exchange chromatography (Waters). The cation-exchange (CEX) column used was a ProPac WCX-10 (Dionex, Sunnyvale, CA, USA). Acidic charge variants were defined as represented by the several antibody peaks that were eluted earlier than the main peak during CEX analysis. In contrast, basic charge variants were defined as represented by the several antibody peaks that were eluted later than the main peak during CEX. Screening of process parameters by PlacketteBurman design PlacketteBurman designs are used to screen the main factors from a large number of process factors (18). These designs can be very useful in preliminary studies in which the principal objective is to identify those factors that can be fixed or excluded in a further optimization process. The variables chosen were initial viable cell density, pH, temperature shift, feed rate, feed timing, initial medium concentration, osmolality, and dissolved oxygen. The variables were selected by a failure modes and effects analysis (FMEA) (Table 1). The definitions of the assessment factorsdseverity of impact, probability of occurrence, and likelihood of detectiondused for FMEA were developed by some of our study group (19). Table 2 lists the factors investigated and the values of each factor used in the experimental design, and Table 3 presents the design matrix. All cell culture was performed in a 250-mL BCC bioreactor. The duration of cell production culture was 14 days. Statistical analysis Statistical tests and design of experiments analysis were performed with JMP 9.0 software (SAS Institute, Cary, NC, USA). Where appropriate, the two-tailed Student’s t-test was performed to determine the statistical significance. Statistical significance was defined as P < 0.05.

TABLE 1. Results of failure mode and effects analysis of the production culture step. Process parameter (unit)

Set point (current process)

Characterization range

S

O

D

RPN

IVCD (105 cells/mL) pH () Temperature shift ()

2.0 6.9  0.02 Not conducted

7 7 7

2 2 2

5 3 3

70 42 42

Feed rate (mL/L) Feed timing (day) Initial medium concentration () Feed medium osmolality (mOsm/kg) DO (%) Temperature ( C) Cell age (day) Initial glucose concentration (g/L) Agitation speed of 1-L scale (rpm) Working volume ()

24.0 3 1.0

1.0e3.0 6.8e7.0 Not conductede 33 C, day 5 12.0e36.0 2e4 0.5e1.5

4 4 4

2 2 2

5 5 3

40 40 24

1000  30

>1000

4

2

3

24

40  2 37.0  0.1 e 4.6

10e60 36.5e37.5 <120 4.0e6.0

4 7 2 4

2 2 1 2

3 1 5 1

24 14 10 8

80

60e100

2

2

1

4

1.0

0.8e1.2

2

2

1

4

DO, dissolved oxygen; IVCD, initial viable cell density; S, severity of impact score; O, probability of occurrence score; D, likelihood of detection score; RPN, risk priority number.

probability of occurrence, and likelihood of detection. The RPN threshold, determined by internal experts, was set at 20. Initial viable cell density, pH, temperature shift, feed rate, feed timing, initial medium concentration, osmolality, and dissolved oxygen leveldeach of which had an RPN of over 20dwere selected. By using a 12-run PlacketteBurman design, we analyzed these eight factors for their effects on the content of acidic charge variants. The factors were tested simultaneously by shifting each of them from a low value (1) to a high value (þ1) (Table 2); among the 12 runs, various combinations of factor levels (1 or þ1) were designated according to the PlacketteBurman design (Table 3). One of the test values (e1 or þ1) was taken as the set point for this process, whereas the other test value was used as the upper or lower limit of the characterization range of FMEA. Regarding selection of the upper or lower limit, we chose the limit that we expected would have the more positive effect on the mAb titer and quality attributes. For each run, the content of acidic charge variants was measured (Table 3, last column). JMP analysis of the results showed that only temperature shift had a significant effect on the content of acidic charge variants (P < 0.05) (Table 4). Temperature shift was thus a key process factor and was selected for further investigation.

Effect of shift temperature on acidic charge variant content Culture was initiated at a density of 2.0  105 cells/mL at 37 C in a 1-L bioreactor. The control conditions were culture at 37 C from beginning to end. To determine the effect of temperature shift on the CHO cell growth, mAb titer, and acidic charge variant content, on day 5 the temperature was shifted to one of two

RESULTS TABLE 2. Details of factors selected for the experimental design.

Screening of process parameters by using PlacketteBurman design FMEA is one of the most common and useful tools for risk analysis in pharmaceutical process development (20e22). We used FMEA to analyze and evaluate the risks of all of the process parameters for mAb titer and quality attributes in the production culture. Each process parameter was assessed in terms of three factorsdseverity of impact, probability of occurrence, and likelihood of detectiondon the basis of established scientific knowledge and our experimental knowledge of mAb titers and quality attributes (Table 1). Each risk priority number (RPN) was calculated by multiplying the scores for severity of impact,

Factor

pH IVCD Temperature shift Feed rate Feed timing Initial medium concentration Feed medium osmolality DO

Units

e 105 cells/mL e mL/h day e mOsm/kg %

Level 1

þ1

6.8 2.0 33 C, day5 0.1 2.0 1 1000 40

6.9 3.0 Not conducted 0.15 3.0 1.5 1200 60

DO, dissolved oxygen. IVCD, initial viable cell density.

Please cite this article in press as: Kishishita, S., et al., Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.10.028

VOL. xx, 2014

EFFECT OF TEMPERATURE SHIFT ON ACIDIC CHARGE VARIANTS

3

TABLE 3. PlacketteBurman design and resulting contents of acidic charge variants. No.

pH

IVCD

Temperature shift

Feed rate

Feed timing

Initial medium concentration

Osmolality of feed medium

DO

Acidic charge variants (%)

1 2 3 4 5 6 7 8 9 10 11 12

þ1 1 1 þ1 1 1 1 þ1 þ1 þ1 1 þ1

þ1 þ1 1 1 þ1 1 1 1 þ1 þ1 þ1 1

þ1 1 þ1 1 1 þ1 1 1 1 þ1 þ1 þ1

þ1 þ1 1 þ1 1 1 þ1 1 1 1 þ1 þ1

þ1 þ1 þ1 1 þ1 1 1 þ1 1 1 1 þ1

þ1 þ1 þ1 þ1 1 þ1 1 1 þ1 1 1 1

þ1 1 þ1 þ1 þ1 1 þ1 1 1 þ1 1 1

þ1 1 1 þ1 þ1 þ1 1 þ1 1 1 þ1 1

33.9 25.4 30.7 22.5 22.3 32.4 18.7 21.6 22.1 31.6 31.7 31.7

DO, dissolved oxygen. IVCD, initial viable cell density.

B 4.0 3.0

33°C 35°C Control

2.0

Shift timing

150 120

*

90 60

33°C 35°C Control

Shift timing

30

* *

* * 1.0 0.0

0 0

2

4 6 8 10 12 14 Culture time (Days)

C Acidic charge variant content on day 14 (%)

mAb titer (g/L)

VCD (105 cells/mL)

A 180

0

2

4 6 8 10 12 14 Culture time (Days)

*

50

*

40 30 20 10 0 33°C

35°C

Control

FIG. 1. Effects of different shift temperatures on cell growth, mAb titer, and acidic charge variant content. (A) Viable cell density (VCD). (B) mAb titer. (C) Content of acidic charge variants on day 14. All data points in panels A to C correspond to the averages of biological triplicates  standard deviation. *P < 0.05 (two-tailed Student’s t-test).

different temperatures, namely 33 C or 35 C. These temperature shifts were maintained until the end of culture. We then examined the cell growth profiles (Fig. 1A). From day 10 onward, reducing the temperature maintained greater numbers of

TABLE 4. Statistical analysis results for PlacketteBurman design. Factors Intercept pH IVCD Temperature shift Feed rate Feed timing Initial medium concentration Osmolality DO

Estimate

t-ratio

Probability > jtj

27.050 0.183 0.783 4.950 0.267 0.550 0.783 0.493 0.350

70.08 0.47 2.03 12.82 0.69 1.42 2.03 1.12 0.91

<0.0001a 0.6672 0.1354 0.001a 0.5393 0.2494 0.1354 0.3433 0.4314

DO, dissolved oxygen. IVCD, initial viable cell density. a Statistically significant (P < 0.05).

viable cells than in the control culture. On day 14, the viable cell density in the controls was 65  105 cells/mL. In contrast, on day 14, at 35 C the viable cell concentration was 98  105 cells/mL and at 33 C it was 118  105 cells/mL. We also examined the effect of temperature shift on product accumulation (Fig. 1B). The maximum mAb titer under control conditions was 3.5 g/L, on day 14. Shifting the temperature to 35 or 33 C resulted in antibody concentrations of 3.1 and 2.8 g/L, respectively. Lowering the culture temperature did not increase mAb production. We determined the contents of acidic charge variants at the different culture temperatures (Fig. 1C). On day 14, the contents of acidic charge variants under control conditions and at 35 C and 33 C were 40%, 30%, and 22%, respectively. There was a significant difference in content between the control conditions and a reduced culture temperature of 35 C or 33 C (P < 0.05, two-tailed Student’s t-test). The lowest level of acidic charge variants was achieved at 33 C, and an approximately 46% reduction was achieved by

Please cite this article in press as: Kishishita, S., et al., Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.10.028

KISHISHITA ET AL.

J. BIOSCI. BIOENG.,

lowering the culture temperature from 37 C to 33 C. Accordingly, lowering the culture temperature could reduce acidic charge variant levels, as expected from the results of the PlacketteBurman design. The content of each charge variant on day 14 is shown in Fig. S1. The content of main peak was increased by the reduction in acidic charge variant content. Effect of timing of temperature shift on acidic charge variant content Culture was initiated at a density of 2.0  105 cells/mL at 37 C in a 1-L bioreactor. The control culture was maintained at 37 C until the end of culture. We examined the effect of temperature shift timing on CHO cells with respect to cell growth, mAb titer, and acidic charge variant content. Temperature shifts were made at two different times, namely on day 3 or 5. The shift temperature was 33 C, and after the shift this temperature was maintained until the end of culture. We first examined the viable cell concentration profiles (Fig. 2A). In the control culture the maximum viable cell concentration was 167  105 cells/mL on day 7. In contrast, the maximum viable cell concentration with a shift to 33 C on day 5 was 168  105 cells/mL on day 7, whereas that with a shift on day 3 was 91  105 cells/mL on day 10. However, although the cells subjected to the early temperature shift grew more slowly than those subjected to control conditions or to a later temperature drop, their density plateaued at day 7 and was still at about the same level at the end of culture. We then evaluated the effect of the temperature shift timing on product accumulation (Fig. 2B). The maximum mAb concentration under control conditions was 3.5 g/L, on day 14. A temperature shift to 33 C on day 5 gave a maximum mAb concentration of 2.8 g/L, on day 14, whereas that on day 3 gave an mAb concentration of 1.8 g/L, on day 14. mAb concentrations were decreased by accelerating the shift timing. Next, we examined the changes in acidic charge variant contents with different timings of the temperature shift to 33 C (Fig. 2C). On

DISCUSSION When process changes are made during mAb productivity optimization, minimizing changes in quality attributes is critical for establishing comparability of therapeutic mAbs. During process development to enhance mAbs productivity, we have sometimes observed increases in the acidic charge variant content. Our goal here was therefore to identify the process parameters affecting the acidic charge variant levels of mAbs expressed in CHO cells. The first step toward our goal was to select factors (i.e., process parameters in the production culture) and to assess the risk posed by each of them to mAb titers and quality attributes. The FMEA exercise enabled us to eliminate process parameters with low RPN scores from further study. We showed here that PlacketteBurman designs could be useful for minimizing the number of runs required in the initial screening of numerous potentially influential process parameters. The resulting data revealed a clear effect of temperature shift on the acidic charge variant content (Table 4). González-Leal et al. (23) proposed that PlacketteBurman designs can be used for media optimization in CHO cell culture. The use of PlacketteBurman

B

A 200 VCD (105 cells/mL)

day 14, the acidic charge variant content in the control was 40%. In contrast, the contents after temperature shifts on days 3 and 5 were 20% and 22%, respectively. Although bringing forward the temperature shift timing from day 5 to day 3 did not further suppress the acidic charge variant content, in both cases the acidic charge variant content was markedly lower with the 33 C temperature shift than in the controls. There was a significant difference in the content between the control conditions and those in which the culture temperature was lowered on day 3 or 5 to 33 C (P < 0.05, two-tailed Student’s t-test). The content of each type of charge variant on day 14 is shown in Fig. S2. The content of main peak was increased by the reduction in acidic charge variant content.

4.0 Day3 Day5 Control

175 Shift timing

150 125 100

* **

75 50 25 0

Day3 Day5 Control

* *

3.0 2.0

2

4 6 8 10 12 14 Culture time (Days)

C

*

Shift timing

*

1.0

*

* *

0.0 0

Acidic charge variant content on day 14 (%)

* *

*

mAb titer (g/L)

4

0

2

4 6 8 10 12 14 Culture time (Days)

*

50

*

40 30 20 10 0 Day 3

Day 5

Control

FIG. 2. Effects of changes in the timing of temperature shift on cell growth, mAb titer, and acidic charge variant content. (A) Viable cell density (VCD). (B) mAb titer. (C) Content of acidic charge variants on day 14. All data points in panels A to C correspond to the averages of biological triplicates  standard deviation. *P < 0.05 (two-tailed Student’s t-test).

Please cite this article in press as: Kishishita, S., et al., Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.10.028

VOL. xx, 2014

EFFECT OF TEMPERATURE SHIFT ON ACIDIC CHARGE VARIANTS

designs provides a rational approach that could save time and resources. Temperature shifts have effects on quality attributes such as glycosylation (24) and aggregation (25). We found here, for the first time to our knowledge, that temperature downshifts could also significantly suppress the levels of acidic charge variants. The acidic charge variant content was reduced by lowering the temperature (Fig. 1) and accelerating the temperature shift timing (Fig. 2). A trend toward reduction in acidic charge variant content by temperature downshift was also observed in another cell line (data not shown). These results showed that temperature downshift was a good way of controlling the acidic charge variant content with the aim of achieving comparability among mAbs. We obtained the same results from temperature downshift at a 1000-L bioreactor scale (data not shown), and we intend to apply this process at the commercial scale in the near future. We found here that mAb production was decreased by temperature downshifts. However, many reports have focused on the beneficial effects of temperature shifts on cell viability and recombinant protein production (26e32). The advantages of using low culture temperatures include maintaining high viability for a longer culture time and enhancing specific protein productivity. Therefore, to clarify the wide applicability of the reduction in acidic charge variant content produced by downshifting the temperature, we need to determine whether such an effect can be obtained in cell lines in which productivity is enhanced by temperature shift. The mechanism by which the content of acidic charge variants is reduced by a temperature downshift has not yet been elucidated. Several reports have found that acidic charge variants are generated by a number of modifications, such as sialic acid (33), glycation (34), non-reducible species (35), reduced disulfide bonds (35), or deamidation in the Fc region (36). To elucidate the mechanism we intend to try to characterize the kinds of modification that are affected by the temperature downshift; we will report the results in the not too distant future. In conclusion, our results indicate that PlacketteBurman designs are useful for the initial screening of bioprocess parameters and that a temperature downshift markedly reduces the levels of acidic peak variants. Further studies are needed to determine whether temperature shifts are widely applicable to the control of acidic charge variant levels and to elucidate the mechanism by which this control occurs. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2014.10.028.

6. 7. 8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18. 19.

20.

21.

22.

ACKNOWLEDGMENT The authors wish to thank all the members of the Chugai Pharmaceutical team for their support during this work. References 1. Dinnis, D. M. and James, D. C.: Engineering mammalian cell factories for improved recombinant monoclonal antibody production: lessons from nature, Biotechnol. Bioeng., 91, 180e189 (2005). 2. Yu, M., Hu, Z. L., Pacis, E., Vijayasankaran, N., Shen, A., and Li, F.: Understanding the intracellular effect of enhanced nutrient feeding toward high titer antibody production process, Biotechnol. Bioeng., 108, 1078e1088 (2011). 3. Arunakumari, A., Dai, X. P., Goldstein, J., Kloth, C., Ghebremariam, H., Macisaac, G., and Wagner, M.: The impact of cell culture medium on cell line and process development timelines and strategies, Biopharm. Int. Suppl., (Jun 2, 2009). 4. Jayapal, K. R., Wlaschin, K. F., Hu, W. S., and Yap, M. G. S.: Recombinant protein therapeutics from CHO cells e 20 years and counting, Chem. Eng. Prog., 103, 40e47 (2007). 5. Harris, R. J., Kabakoff, B., Macchi, F. D., Shen, F. J., Kwong, M., Andya, J. D., Shire, S. J., Bjork, N., Totpal, K., and Chen, A. B.: Identification of multiple

23.

24.

25.

26.

27.

28.

5

sources of charge heterogeneity in a recombinant antibody, J. Chromatogr. B Biomed. Sci. Appl., 752, 233e245 (2001). Liu, H., Gaza-Bulseco, G., Faldu, D., Chumsae, C., and Sun, J.: Heterogeneity of monoclonal antibodies, J. Pharm. Sci., 97, 2426e2447 (2008). Vlasak, J. and Ionescu, R.: Heterogeneity of monoclonal antibodies revealed by charge-sensitive methods, Curr. Pharm. Biotechnol., 9, 468e481 (2008). Triguero, D., Buciak, J. B., Yang, J., and Pardridge, W. M.: Blood-brain barrier transport of cationized immunoglobulin G: enhanced delivery compared to native protein, Proc. Natl. Acad. Sci. USA, 86, 4761e4765 (1989). Pardridge, W. M., Kang, Y. S., Diagne, A., and Zack, J. A.: Cationized hyperimmune immunoglobulins: pharmacokinetics, toxicity evaluation and treatment of human immunodeficiency virus-infected human-peripheral blood lymphocytes-severe combined immune deficiency mice, J. Pharmacol. Exp. Ther., 276, 246e252 (1996). Hong, G., Bazin-Redureau, M. I., and Scherrmann, J. M.: Pharmacokinetics and organ distribution of cationized colchicine-specific IgG and Fab fragments in rat, J. Pharm. Sci., 88, 147e153 (1999). Rodwell, J. D., Alvarez, V. L., Lee, C., Lopes, A. D., Goers, J. W., King, H. D., Powsner, H. J., and McKearn, T. J.: Site-specific covalent modification of monoclonal antibodies: in vitro and in vivo evaluations, Proc. Natl. Acad. Sci. USA, 83, 2632e2636 (1986). Gangopadhyay, A., Petrick, A. T., and Thomas, P.: Modification of antibody isoelectric point affects biodistribution of 111-indium-labeled antibody, Nucl. Med. Biol., 23, 257e261 (1996). Khawli, L. A., Mizokami, M. M., Sharifi, J., Hu, P., and Epstein, A. L.: Pharmacokinetic characteristics and biodistribution of radioiodinated chimeric TNT-1, -2, and -3 monoclonal antibodies after chemical modification with biotin, Cancer Biother Radiopharm., 17, 359e370 (2002). Lee, H. J. and Pardridge, W. M.: Monoclonal antibody radiopharmaceuticals: cationization, pegylation, radiometal chelation, pharmacokinetics, and tumor imaging, Bioconjug Chem., 14, 546e553 (2003). Perera, R. M., Zoncu, R., Johns, T. G., Pypaert, M., Lee, F. T., Mellman, I., Old, L. J., Toomre, D. K., and Scott, A. M.: Internalization, intracellular trafficking, and biodistribution of monoclonal antibody 806: a novel antiepidermal growth factor receptor antibody, Neoplasia, 9, 1099e1110 (2007). Pallavicini, M. G., DeTeresa, P. S., Rosette, C., Gray, J. W., and Wurm, F. M.: Effects of methotrexate on transfected DNA stability in mammalian cells, Mol. Cell. Biol., 10, 401e404 (1990). Gandor, C., Leist, C., Fiechter, A., and Asselbergs, F. A.: Amplification and expression of recombinant genes in serum-independent Chinese hamster ovary cells, FEBS Lett., 377, 290e294 (1995). Plackett, R. L. and Burman, J. P.: The design of optimum multifactorial experiments, Biometals, 33, 305e325 (1946). Nagashima, H., Watari, A., Shinoda, Y., Okamoto, H., and Takuma, S.: Application of a quality by design approach to the cell culture process of monoclonal antibody production, resulting in the establishment of a design space, J. Pharm. Sci., 102, 4274e4283 (2013). Abu-Absi, S. F., Yang, L., Thompson, P., Jiang, C., Kandula, S., Schilling, B., and Shukla, A. A.: Defining process design space for monoclonal antibody cell culture, Biotechnol. Bioeng., 106, 894e905 (2010). Looby, M., Ibarra, N., Pierce, J. J., Buckley, K., O’Donovan, E., Heenan, M., Moran, E., Farid, S. S., and Baganz, F.: Application of quality by design principles to the development and technology transfer of a major process improvement for the manufacture of a recombinant protein, Biotechnol. Prog., 27, 1718e1729 (2011). Rouiller, Y., Solacroup, T., Deparis, V., Barbafieri, M., Gleixner, R., Broly, H., and Eon-Duval, A.: Application of quality by design to the characterization of the cell culture process of an Fc-Fusion protein, Eur. J. Pharm. Biopharm., 81, 426e437 (2012). González-Leal, I. J., Carrillo-Cocom, L. M., Ramírez-Medrano, A., LópezPacheco, F., Bulnes-Abundis, D., Webb-Vargas, Y., and Alvarez, M. M.: Use of a Plackett-Burman statistical design to determine the effect of selected amino acids on monoclonal antibody production in CHO cells, Biotechnol. Prog., 27, 1709e1717 (2011). Borys, M. C., Dalal, N. G., Abu-Absi, N. R., Khattak, S. F., Jing, Y., Xing, Z., and Li, Z. J.: Effects of culture conditions on N-glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells, Biotechnol. Bioeng., 105, 1048e1057 (2010). Gomez, N., Subramanian, J., Ouyang, J., Nguyen, M. D., Hutchinson, M., Sharma, V. K., Lin, A. A., and Yuk, I. H.: Culture temperature modulates aggregation of recombinant antibody in CHO cells, Biotechnol. Bioeng., 109, 125e136 (2012). Fox, S. R., Patel, U. A., Yap, M. G., and Wang, D. I.: Maximizing interferon-gamma production by Chinese hamster ovary cells through temperature shift optimization: experimental and modeling, Biotechnol. Bioeng., 85, 177e184 (2004). Furukawa, K. and Ohsuye, K.: Enhancement of productivity of recombinant alpha-amidating enzyme by low temperature culture, Cytotechnology, 31, 85e94 (1999). Hendrick, V., Winnepenninckx, P., Abdelkafi, C., Vandeputte, O., Cherlet, M., Marique, T., Renemann, G., Loa, A., Kretzmer, G., and Werenne, J.: Increased

Please cite this article in press as: Kishishita, S., et al., Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.10.028

6

29.

30.

31.

32.

KISHISHITA ET AL. productivity of recombinant tissular plasminogen activator (t-PA) by butyrate and shift of temperature: a cell cycle phases analysis, Cytotechnology, 36, 71e83 (2001). Kaufmann, H., Mazur, X., Fussenegger, M., and Bailey, J. E.: Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells, Biotechnol. Bioeng., 63, 573e582 (1999). Trummer, E., Fauland, K., Seidinger, S., Schriebl, K., Lattenmayer, C., Kunert, R., Vorauer-Uhl, K., Weik, R., Borth, N., Katinger, H., and Müller, D.: Process parameter shifting: Part II. Biphasic cultivationda tool for enhancing the volumetric productivity of batch processes using Epo-Fc expressing CHO cells, Biotechnol. Bioeng., 94, 1045e1052 (2006). Chen, Z.: Temperature shift as a process optimization step for the production of pro-urokinase by a recombinant Chinese hamster ovary cell line in high-density perfusion culture, J. Biosci. Bioeng., 97, 239e243 (2004). Yoon, S. K., Ahn, Y.-H., and Jeong, M. H.: Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor, Appl. Microbiol. Biotechnol., 76, 83e89 (2007).

J. BIOSCI. BIOENG., 33. Zhang, T., Bourret, J., and Cano, T.: Isolation and characterization of therapeutic antibody charge variants using cation exchange displacement chromatography, J. Chromatogr. A, 1218, 5079e5086 (2011). 34. Miller, A. K., Hambly, D. M., Kerwin, B. A., Treuheit, M. J., and Gadgil, H. S.: Characterization of site-specific glycation during process development of a human therapeutic monoclonal antibody, J. Pharm. Sci., 100, 2543e2550 (2011). 35. Khawli, L. A., Goswami, S., Hutchinson, R., Kwong, Z. W., Yang, J., Wang, X., Yao, Z., Sreedhara, A., Cano, T., Tesar, D., and other 8 authors: Charge variants in IgG1: isolation, characterization, in vitro binding properties and pharmacokinetics in rats, MAbs, 2, 613e624 (2010). 36. Gandhi, S., Ren, D., Xiao, G., Bondarenko, P., Sloey, C., Ricci, M. S., and Krishnan, S.: Elucidation of degradants in acidic peak of cation exchange chromatography in an IgG1 monoclonal antibody formed on long-term storage in a liquid formulation, Pharm. Res., 29, 209e224 (2012).

Please cite this article in press as: Kishishita, S., et al., Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.10.028