**3. Structural heterogeneity of recombinant human antibody light chains (including catalytic light chains)**

### **3.1. Phenomena observed in several human antibody light chains**

#### *3.1.1. Chromatograms of antibody light chain C51*

In this section, we will describe the phenomena of structural diversity using full-length light chains of human antibodies. **Figure 2a** shows the amino acid sequences of human antibody kappa light chains of C51, #4 and #7. The proteins were expressed in *Escherichia coli* in accordance with the protocol described in the section of Materials and Methods in Refs. [19, 31, 32]. Methionine was adducted at the N-terminus and confirmed by amino acid sequence analysis after cloning the cDNA of the light chain into the *Nco I* site of the pET-20b vector. Leu and Glu residues were inserted by employing the *Xho I* site before a histidine–tag (His × 6) included in the vector for purification.

After the transformed *E. coli* cells were recovered by centrifugation, they were sonicated. Then, the soluble fraction was subjected to purification using Ni-NTA affinity chromatography. The result of a Ni-NTA affinity chromatography for the C51 light chain is shown in **Figure 2b**. The C51 light chain was eluted from fraction 28 (Fr.28) to Fr.44. Fr.35 showed the maximum absorbance. Fr.35 was collected and analyzed by SDS-PAGE with CBB staining, where the C51 light chain was mainly the monomer form with a slight contamination of dimer forms.

*3.1.2. Effect of copper ions*

(Cu2+) was added to either the cell suspension after recovery of the cells (cell-

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suspension) or the eluent of Ni-NTA chromatography (Ni-NTA eluent). By the addition of Cu2+ to the cell suspension, the chromatogram changed to that shown in **Figure 2d**, where a main peak (peak 4) was observed at the retention time of 26 min but other peaks (1, 2 and 3) were small. The SDS-PAGE gave a dimer for peak 4. In **Figure 2e**, 20 μM of Cu2+ (0.5 equivalent to the light chain)

detected. The SDS-PAGE gave a dimer for peak 3. The addition of Cu2+ led to formation of dimers.

**Figure 2.** C51 light chain. (a) Amino acid sequences of human light chains (kappa type). (b) First-step purification of C51 light chain. (b-1) Steps from *E. coli* culture to Ni-NTA column chromatography. (b-2) Ni-NTA column chromatogram for C51 light chain. (b-3) Results of SDS-PAGE of fraction 28–45. C51 light chain was mainly the monomer form with a slight contamination of dimer forms at approximately 45 kDa. (c) Cation exchange chromatography as the secondstep purification without copper ion. Several peaks were observed from 15 to 27 min. The peaks 1, 2, and 3 were the monomer, the mixture of monomer and dimer, and the dimer, respectively. (d) Cation exchange chromatography as the second-step purification with addition of 0.5 eq. copper ion in cell suspension. A main peak (peak 4) was observed at the retention time of 26 min but other peaks (1–3) were small. The SDS-PAGE gave a dimer for peak 4. (e) Cation exchange chromatography as the second-step purification with addition of 0.5 eq. copper ion in the Ni-NTA eluent. Only a main peak (peak 3) was observed at the retention time of 26 min but other peaks (1 and 2) were scarcely

Twenty μM of CuCl<sup>2</sup>

The eluted fractions from Fr.30 to Fr.40 were collected and subjected to a cation exchange chromatography. **Figure 2c** shows the chromatogram, where several peaks were observed. The SDS-PAGEs of the peaks were shown in the figure on the right side. The peaks 1, 2, and 3 were the monomer, the mixture of monomer and dimer, and the dimer, respectively.

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**Figure 2.** C51 light chain. (a) Amino acid sequences of human light chains (kappa type). (b) First-step purification of C51 light chain. (b-1) Steps from *E. coli* culture to Ni-NTA column chromatography. (b-2) Ni-NTA column chromatogram for C51 light chain. (b-3) Results of SDS-PAGE of fraction 28–45. C51 light chain was mainly the monomer form with a slight contamination of dimer forms at approximately 45 kDa. (c) Cation exchange chromatography as the secondstep purification without copper ion. Several peaks were observed from 15 to 27 min. The peaks 1, 2, and 3 were the monomer, the mixture of monomer and dimer, and the dimer, respectively. (d) Cation exchange chromatography as the second-step purification with addition of 0.5 eq. copper ion in cell suspension. A main peak (peak 4) was observed at the retention time of 26 min but other peaks (1–3) were small. The SDS-PAGE gave a dimer for peak 4. (e) Cation exchange chromatography as the second-step purification with addition of 0.5 eq. copper ion in the Ni-NTA eluent. Only a main peak (peak 3) was observed at the retention time of 26 min but other peaks (1 and 2) were scarcely detected. The SDS-PAGE gave a dimer for peak 3. The addition of Cu2+ led to formation of dimers.

#### *3.1.2. Effect of copper ions*

**2.2. Recombinant monoclonal antibody Herceptin**

234 Antibody Engineering

capillary isoelectric focusing and 2D-gel electrophoresis.

reaction of the deamidation and the heterogeneity is lost.

**3.1. Phenomena observed in several human antibody light chains**

**chains (including catalytic light chains)**

*3.1.1. Chromatograms of antibody light chain C51*

included in the vector for purification.

The structural diversity of antibodies has been found about 20 years ago [26, 27], suggesting that an antibody has some different structures (not a mono-form structure) caused by the various electrical charges. Regarding the charge heterogeneity, Harris et al. [24] and Nebija et al. [25, 30] have extensively studied this phenomenon with recombinant antibodies using

The latter papers showed multiple spots of pI spreading at heavy and light chains, caused by generation of charge-related isoforms. The pI spreading pattern in 2D-gel electrophoresis [30] is similar to our cases. The effects of sugar chains are also taken into account for molecular heterogeneity. As it is well known that an antibody light chain has no sugar chain, this can be excluded for this subunit. Thus, in our case, it is thought that the structural diversity is mostly due to the heterogeneity of the different electrical charges. In addition, the possibility of deamidation is excluded because it is hardly considered that the addition of a copper ion causes a reverse

The former paper [24] pointed out a deamidation of Asn residues in the protein.

**3. Structural heterogeneity of recombinant human antibody light** 

In this section, we will describe the phenomena of structural diversity using full-length light chains of human antibodies. **Figure 2a** shows the amino acid sequences of human antibody kappa light chains of C51, #4 and #7. The proteins were expressed in *Escherichia coli* in accordance with the protocol described in the section of Materials and Methods in Refs. [19, 31, 32]. Methionine was adducted at the N-terminus and confirmed by amino acid sequence analysis after cloning the cDNA of the light chain into the *Nco I* site of the pET-20b vector. Leu and Glu residues were inserted by employing the *Xho I* site before a histidine–tag (His × 6)

After the transformed *E. coli* cells were recovered by centrifugation, they were sonicated. Then, the soluble fraction was subjected to purification using Ni-NTA affinity chromatography. The result of a Ni-NTA affinity chromatography for the C51 light chain is shown in **Figure 2b**. The C51 light chain was eluted from fraction 28 (Fr.28) to Fr.44. Fr.35 showed the maximum absorbance. Fr.35 was collected and analyzed by SDS-PAGE with CBB staining, where the C51 light chain was mainly the monomer form with a slight contamination of dimer forms.

The eluted fractions from Fr.30 to Fr.40 were collected and subjected to a cation exchange chromatography. **Figure 2c** shows the chromatogram, where several peaks were observed. The SDS-PAGEs of the peaks were shown in the figure on the right side. The peaks 1, 2, and 3

were the monomer, the mixture of monomer and dimer, and the dimer, respectively.

Twenty μM of CuCl<sup>2</sup> (Cu2+) was added to either the cell suspension after recovery of the cells (cellsuspension) or the eluent of Ni-NTA chromatography (Ni-NTA eluent). By the addition of Cu2+ to the cell suspension, the chromatogram changed to that shown in **Figure 2d**, where a main peak (peak 4) was observed at the retention time of 26 min but other peaks (1, 2 and 3) were small. The SDS-PAGE gave a dimer for peak 4. In **Figure 2e**, 20 μM of Cu2+ (0.5 equivalent to the light chain) was added to the Ni-NTA eluent. In this case, only a main peak (peak 3) was observed at the retention time of 26 min but other peaks (1 and 2) were scarcely detected. The SDS-PAGE gave a dimer for peak 3. The addition of Cu2+ led to dimer formation.

Mass spectroscopic (MS) analysis was performed for the main peaks observed above (data not shown). Briefly, a monomeric light chain was detected at 25,000 m/z and a dimer at 49,000 m/z. A small trimer and tetramer were also detected at 74,000 and 98,000 m/z, respectively. By the addition of Cu2+ to the cell suspension or the Ni-NTA eluent, the signal for the monomer was substantially reduced. Conclusively, the addition of Cu2+ was effective for the formation of the dimer.

In order to examine the pI of the light chain, 2D-gel electrophoresis was performed with and without the addition of Cu2+. The results are shown in **Figure 3a** and **b**. In the case without Cu2+, many spots at different pIs were observed with the same molecular size of 31 kDa (**Figure 3a**). The pI spots were widely located from 6.12 to 10.0. The strong spot was observed at pI = 6.45– 6.73. In contrast, in the case with the addition of Cu2+, the spots were gathered on the strongest spot at pI = 6.57, while two faint spots were detected at around pI = 6.32 and 6.90 (**Figure 3b**). It is evident that the electrical charges of the molecule became mono-form by the effect of Cu2+.

#### **3.2. #4 and #7 light chains**

It must be invested whether or not the changes from multi-molecular forms to mono-molecular forms by the addition of copper ions is a general phenomenon. The following experiments were carried out.

#### *3.2.1. Chromatograms*

For the purpose of confirmation of the observed phenomena on the structural diversity, other antibody light chains such as #4 and #7 were examined. As stated above, the chromatogram in Ni-NTA purification is similar for many light chains. Thus, the following is focused on the results of cation exchange chromatography, which were very different in each light chain used. The effect of copper ions on the diversity issue will be discussed.

The cation chromatograms for #4 light chain are shown in the cases without and with Cu2+ as presented in **Figure 4a** and **b**, respectively. In the case without Cu2+, there were several peaks. The results of SDS-PAGE (non-reduced condition) corresponding to three peaks are also shown. The observed peaks were a mixture of monomers and dimers. Namely, several structurally different light chains caused by different electrical charges are coexisting in the solution. However, when 20 μM of Cu2+ (0.5 equivalent to the light chain) was added to the Ni-NTA eluent, the several peaks in **Figure 4a** surprisingly became a single peak (**Figure 4b**), which was mainly the dimer.

When 15 μM of Cu2+ (0.38 eq.) was added to the Ni-NTA eluent, two peaks at 12 min and 23 min of retention time were observed in the chromatography. The results of SDS-PAGE analysis (**Figure 5b**) indicated that both peak A and peak B were dimers. It is interesting that two kinds of dimers with different electrical charges were coexisting. When 40 μM of Cu2+ (1.0 eq.) was added to the same eluent, only peak C, which corresponds to peak A in **Figure 5b**, was observed at 12 min and peak B was not detected (**Figure 5c**). It is thought that peak B moved to peak A in **Figure 5b**. Conclusively, the dimeric light chains possessing two kinds of electrical charges became one kind of state possessing a unique electrical charge by the addition of 40 μM of Cu2+.

**Figure 3.** 2D-gel electrophoresis for C51 light chain. (a) Without copper ion. The pI spots were widely located from 6.12 to 10.0. (b) With copper ion. The spots were gathered on the strongest spot at pI = 6.57, although two faint spots were

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detected at around pI = 6.32 and 6.90.

In the case of the chromatography for #7 light chain, huge three peaks were observed in the case without Cu2+ as shown in **Figure 5a**. The peak of the retention time at 9 min was the monomer. The peak at 13 min was a mixture of monomer and dimer. The peak at 22 min was also a mixture. Note that light chains possess different molecular sizes and electrical charges in solution.

was added to the Ni-NTA eluent. In this case, only a main peak (peak 3) was observed at the retention time of 26 min but other peaks (1 and 2) were scarcely detected. The SDS-PAGE gave a

Mass spectroscopic (MS) analysis was performed for the main peaks observed above (data not shown). Briefly, a monomeric light chain was detected at 25,000 m/z and a dimer at 49,000 m/z. A small trimer and tetramer were also detected at 74,000 and 98,000 m/z, respectively. By the addition of Cu2+ to the cell suspension or the Ni-NTA eluent, the signal for the monomer was substantially reduced. Conclusively, the addition of Cu2+ was effective for the

In order to examine the pI of the light chain, 2D-gel electrophoresis was performed with and without the addition of Cu2+. The results are shown in **Figure 3a** and **b**. In the case without Cu2+, many spots at different pIs were observed with the same molecular size of 31 kDa (**Figure 3a**). The pI spots were widely located from 6.12 to 10.0. The strong spot was observed at pI = 6.45– 6.73. In contrast, in the case with the addition of Cu2+, the spots were gathered on the strongest spot at pI = 6.57, while two faint spots were detected at around pI = 6.32 and 6.90 (**Figure 3b**). It is evident that the electrical charges of the molecule became mono-form by the effect of Cu2+.

It must be invested whether or not the changes from multi-molecular forms to mono-molecular forms by the addition of copper ions is a general phenomenon. The following experiments

For the purpose of confirmation of the observed phenomena on the structural diversity, other antibody light chains such as #4 and #7 were examined. As stated above, the chromatogram in Ni-NTA purification is similar for many light chains. Thus, the following is focused on the results of cation exchange chromatography, which were very different in each light chain

The cation chromatograms for #4 light chain are shown in the cases without and with Cu2+ as presented in **Figure 4a** and **b**, respectively. In the case without Cu2+, there were several peaks. The results of SDS-PAGE (non-reduced condition) corresponding to three peaks are also shown. The observed peaks were a mixture of monomers and dimers. Namely, several structurally different light chains caused by different electrical charges are coexisting in the solution. However, when 20 μM of Cu2+ (0.5 equivalent to the light chain) was added to the Ni-NTA eluent, the several peaks in **Figure 4a** surprisingly became a single peak (**Figure 4b**),

In the case of the chromatography for #7 light chain, huge three peaks were observed in the case without Cu2+ as shown in **Figure 5a**. The peak of the retention time at 9 min was the monomer. The peak at 13 min was a mixture of monomer and dimer. The peak at 22 min was also a mixture. Note that light chains possess different molecular sizes and electrical charges

used. The effect of copper ions on the diversity issue will be discussed.

dimer for peak 3. The addition of Cu2+ led to dimer formation.

formation of the dimer.

236 Antibody Engineering

**3.2. #4 and #7 light chains**

were carried out.

*3.2.1. Chromatograms*

which was mainly the dimer.

in solution.

**Figure 3.** 2D-gel electrophoresis for C51 light chain. (a) Without copper ion. The pI spots were widely located from 6.12 to 10.0. (b) With copper ion. The spots were gathered on the strongest spot at pI = 6.57, although two faint spots were detected at around pI = 6.32 and 6.90.

When 15 μM of Cu2+ (0.38 eq.) was added to the Ni-NTA eluent, two peaks at 12 min and 23 min of retention time were observed in the chromatography. The results of SDS-PAGE analysis (**Figure 5b**) indicated that both peak A and peak B were dimers. It is interesting that two kinds of dimers with different electrical charges were coexisting. When 40 μM of Cu2+ (1.0 eq.) was added to the same eluent, only peak C, which corresponds to peak A in **Figure 5b**, was observed at 12 min and peak B was not detected (**Figure 5c**). It is thought that peak B moved to peak A in **Figure 5b**. Conclusively, the dimeric light chains possessing two kinds of electrical charges became one kind of state possessing a unique electrical charge by the addition of 40 μM of Cu2+.

UV/VIS spectroscopy for these peaks was also conducted. The results are shown in **Figure 6**. We could see the absorbance around 560 nm, which is assigned to the absorbance of the interaction of cupper with the amino acids for peak A and peak C, but not for peak B, whose spec-

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In order to investigate the morphology of antibody light chains, we conducted atomic force microscopy (AFM) analysis. The peaks of A, B, and C were collected and subjected to AFM analysis as shown in **Figure 7a**–**c**. **Figure 7a** shows the AFM image for peak A. The images for peak B and C are shown in **Figure 7b** and **c**, respectively. The red circle represents the clear image of the dimeric light chain. The size of the dimer was roughly estimated at an

**Figure 6.** UV/VIS spectroscopy. The absorbance around 560 nm, which is assigned to the absorbance of the interaction of a copper ion with the amino acids, was observed for peak A and peak C, but not for peak B. It is obvious that peak B

**Figure 7.** AFM analysis. The peaks of A, B, and C were collected and subjected to AFM analysis. The red circle represents the clear image of the dimeric light chain. (a) Peak A included Cu2+ with the ratio of Cu/#7 light chain = 0.48. (b) Peak B did not include Cu2+ (Cu/#7 light chain = 0.03). (c) Peak C included Cu2+ with the ratio of Cu/#7 light chain = 0.64. The size of the dimer was roughly estimated at an approximate length of 20 nm, the width of 10 nm, and the height of 4 nm.

trum was very similar without copper ion.

*3.2.2. AFM analysis*

has no copper ion.

**Figure 4.** Cation exchange chromatography for #4 light chain. (a) Without copper ion. There were mainly three peaks, which were a mixture of monomers and dimers. Namely, several structurally different light chains caused by different electrical charges are coexisting in the solution. (b) With copper ion of 0.5 eq. When 0.5 equivalent to the light chain was added to the Ni-NTA eluent, the several peaks became a single peak of mainly the dimer.

**Figure 5.** Cation exchange chromatography for #7 light chain. (a) Without copper ion. Huge three peaks were observed. The peak of the retention time at 9 min was the monomer, peak at 13 min was the mixture of monomers and dimers, and peak at 22 min was a mixture. In this case, the light chains possess different molecular sizes and electrical charges in solution. (b) With copper ion of 0.38 eq. Two peaks at 12 min and 23 min retention time were observed and both peak A and peak B were dimers. (c) With copper ion of 1.0 eq. Only peak C, which corresponds to peak A in (b), was observed at 12 min as the dimer form.

A chemical analysis of Cu2+ gave interesting results. The ratio of Cu to light chain (Cu/light chain) is 0.48, 0.03, and 0.64 for peaks A, B and C, respectively. When the ratio of Cu/light chain was 0.48 in **Figure 5b** and 0.64 in **Figure 5c**, the dimer was eluted at 12 min. In contrast, at a ratio of 0.03, the retention time was 23 min. These results suggest that whenever enough Cu2+ is present in the solution, an electrically homogeneous light chain could be observed at a retention time of 12 min.

UV/VIS spectroscopy for these peaks was also conducted. The results are shown in **Figure 6**. We could see the absorbance around 560 nm, which is assigned to the absorbance of the interaction of cupper with the amino acids for peak A and peak C, but not for peak B, whose spectrum was very similar without copper ion.

#### *3.2.2. AFM analysis*

A chemical analysis of Cu2+ gave interesting results. The ratio of Cu to light chain (Cu/light chain) is 0.48, 0.03, and 0.64 for peaks A, B and C, respectively. When the ratio of Cu/light chain was 0.48 in **Figure 5b** and 0.64 in **Figure 5c**, the dimer was eluted at 12 min. In contrast, at a ratio of 0.03, the retention time was 23 min. These results suggest that whenever enough Cu2+ is present in the solution, an electrically homogeneous light chain could be observed at a retention time of 12 min.

**Figure 5.** Cation exchange chromatography for #7 light chain. (a) Without copper ion. Huge three peaks were observed. The peak of the retention time at 9 min was the monomer, peak at 13 min was the mixture of monomers and dimers, and peak at 22 min was a mixture. In this case, the light chains possess different molecular sizes and electrical charges in solution. (b) With copper ion of 0.38 eq. Two peaks at 12 min and 23 min retention time were observed and both peak A and peak B were dimers. (c) With copper ion of 1.0 eq. Only peak C, which corresponds to peak A in (b), was observed at 12 min as the dimer form.

**Figure 4.** Cation exchange chromatography for #4 light chain. (a) Without copper ion. There were mainly three peaks, which were a mixture of monomers and dimers. Namely, several structurally different light chains caused by different electrical charges are coexisting in the solution. (b) With copper ion of 0.5 eq. When 0.5 equivalent to the light chain was

added to the Ni-NTA eluent, the several peaks became a single peak of mainly the dimer.

238 Antibody Engineering

In order to investigate the morphology of antibody light chains, we conducted atomic force microscopy (AFM) analysis. The peaks of A, B, and C were collected and subjected to AFM analysis as shown in **Figure 7a**–**c**. **Figure 7a** shows the AFM image for peak A. The images for peak B and C are shown in **Figure 7b** and **c**, respectively. The red circle represents the clear image of the dimeric light chain. The size of the dimer was roughly estimated at an

**Figure 6.** UV/VIS spectroscopy. The absorbance around 560 nm, which is assigned to the absorbance of the interaction of a copper ion with the amino acids, was observed for peak A and peak C, but not for peak B. It is obvious that peak B has no copper ion.

**Figure 7.** AFM analysis. The peaks of A, B, and C were collected and subjected to AFM analysis. The red circle represents the clear image of the dimeric light chain. (a) Peak A included Cu2+ with the ratio of Cu/#7 light chain = 0.48. (b) Peak B did not include Cu2+ (Cu/#7 light chain = 0.03). (c) Peak C included Cu2+ with the ratio of Cu/#7 light chain = 0.64. The size of the dimer was roughly estimated at an approximate length of 20 nm, the width of 10 nm, and the height of 4 nm.

approximate length of 20 nm, the width of 10 nm, and the height of 4 nm. The lateral and height length are comparable with the AFM image of IgG by Querghi et al. [33]. We could not identify the position of the copper ion residing in the light chain from this AFM analysis.
