**2. Structural heterogeneity of monoclonal antibodies**

#### **2.1. Examples found in natural monoclonal antibodies**

We have found an interesting phenomenon in 2D-gel electrophoresis for mouse type monoclonal antibodies (mAbs), which were prepared against the hemagglutinin molecule of influenza virus [29]. In the experiment, these monoclonal antibodies showed many different spots at the same molecular size [28]. **Figure 1a** shows the results using InfA-9 mAb. In the case, the whole antibody and the subunits, heavy and light chains separated and purified from the parent whole antibody, were analyzed by 2D-gel electrophoresis. In **Figure 1a**, many spots in the whole antibody of InfA-9 are shown. The clear four spots (pI = 6.0, 6.1, 6.2 and 6.5) in the heavy chain and three spots (pI = 5.9, 6.1 and 6.5) in the light chain were detected. Except these spots, many faint spots were observed in the same molecular size (the spots observed over the heavy chain were unknown). Then, the heavy and light chains were separated from the whole antibody of InfA-9, highly purified, and submitted to 2D-gel electrophoresis. The results exhibited the similar phenomena. In the heavy chain, similar spots are seen, and clear five spots (pI = 6.1, 6.3, 6.5, 6.7 and 6.9) were detected in this case. The pI positions of the spots

were a little bit different compared to the whole antibody. For the case of the light chain, clear six spots (pI = 5.7, 5.9, 6.0, 6.1, 6.2 and 6.7) were detected. The number of spots increased compared with those of the whole antibody. These phenomena are not exceptional, but general. The mAb, InfA-15, exhibited similar results. **Figure 1b** shows 2D-gel electrophoresis using InfA-15 mAb. In the results of the whole antibody and the heavy chain and the light chain, the pattern of spots were a little different from those of InfA-9 mAb, but several different pI spots were observed in all cases. Namely, different pI spots are present at the same molecular size in any mAb. Note that structural diversity (molecular heterogeneity) should be existing even in the monoclonal antibody and the subunits, while they are a single protein. In our case, it is considered that the various electrical charges of the molecule may be one of the causes.

heterogeneity of antibodies are generally occurring events.

**Figure 1.** 2D electrophoresis for mouse type monoclonal antibodies against hemagglutinin molecule of influenza virus. SDS-PAGE; Running gel 12.5%. Strip; pH 3–10 nonlinear 7 cm. Sample; whole antibody 3.2 μg, heavy and light chain: 1.6 μg. Staining; deep purple (GE Healthcare). (a) InfA-9 monoclonal antibody. Whole antibody: many spots are seen in the whole antibody of InfA-9. The clear four spots (pI = 6.0, 6.1, 6.2 and 6.5) in the heavy chain and three spots (pI = 5.9, 6.1 and 6.5) in the light chain were detected. Heavy chain (H): clear five spots (pI = 6.1, 6.3, 6.5, 6.7 and 6.9) were detected. The pI positions of the spots were a little bit different compared to the whole antibody. Light chain (L): clear six spots (pI = 5.7, 5.9, 6.0, 6.1, 6.2 and 6.7) were detected. The number of spots increased compared with those of the whole antibody. (b) InfA-15 monoclonal antibody. For the whole antibody and the heavy and light chain, similar results showing many spots at different pI for the same molecular sizes were observed, suggesting that the molecular

Structural Diversity Problems and the Solving Method for Antibody Light Chains

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developed [4–14] for the last two decades from the viewpoint of both basic research and the application, where it has been proved that there are many catalytic antibodies being effective against anti-rabies virus [15], anti-influenza virus [16], anti-*Helicobacter pylori* [17], anti-HIV [7, 8, 10], anti-Alzheimer's disease [14, 18], etc. Interestingly, some of them have been advanced to the stage tested *in vivo* in this decade [15–19]. In the case of catalytic antibodies, some of them play the role as a whole structure of IgG [5, 9, 11, 13], IgA [20], or IgM [21–23]. On the other hand, in some cases, their subunits (light chain or heavy chain) exhibit unique functions [1–4, 6–8, 12]. Once the antibody subunits are separated, the structure of the light or heavy chain becomes flexible and has a tendency to possess structural diversity (or molecular heterogeneity). Regarding structural heterogeneity, it was found about 20 years ago that a whole antibody possesses the structural heterogeneity. These studies were extensively studied by Harris et al. [24] and Nebija

et al. [25] using the capillary isoelectric focusing and the 2D-gel electrophoresis [26, 27].

we can best make a significant structure with high reproducibility and productivity.

light chain (CL) plays an important role in generating a mono-molecular form.

**2. Structural heterogeneity of monoclonal antibodies**

**2.1. Examples found in natural monoclonal antibodies**

structure in detail.

232 Antibody Engineering

We have also reported about the molecular heterogeneity caused by different electrical charges and different molecular size in mouse monoclonal antibody [28]. This phenomenon is not good for the preparation efficacy, high reproducibility, and practical application. In addition, the structural diversity leads us to ask what structure plays the most important role in exhibiting the catalytic antibody functions. It also provides us with another subject of how

We have recently found a crucial method to solve the heterogeneity problem by using copper ion, which can convert the multi-molecular forms into mono-molecular forms for many recombinant human antibody light chains. In addition, the constant region domain of the

In this review, we will describe a novel method for preparing a single and defined mono-form

We have found an interesting phenomenon in 2D-gel electrophoresis for mouse type monoclonal antibodies (mAbs), which were prepared against the hemagglutinin molecule of influenza virus [29]. In the experiment, these monoclonal antibodies showed many different spots at the same molecular size [28]. **Figure 1a** shows the results using InfA-9 mAb. In the case, the whole antibody and the subunits, heavy and light chains separated and purified from the parent whole antibody, were analyzed by 2D-gel electrophoresis. In **Figure 1a**, many spots in the whole antibody of InfA-9 are shown. The clear four spots (pI = 6.0, 6.1, 6.2 and 6.5) in the heavy chain and three spots (pI = 5.9, 6.1 and 6.5) in the light chain were detected. Except these spots, many faint spots were observed in the same molecular size (the spots observed over the heavy chain were unknown). Then, the heavy and light chains were separated from the whole antibody of InfA-9, highly purified, and submitted to 2D-gel electrophoresis. The results exhibited the similar phenomena. In the heavy chain, similar spots are seen, and clear five spots (pI = 6.1, 6.3, 6.5, 6.7 and 6.9) were detected in this case. The pI positions of the spots

**Figure 1.** 2D electrophoresis for mouse type monoclonal antibodies against hemagglutinin molecule of influenza virus. SDS-PAGE; Running gel 12.5%. Strip; pH 3–10 nonlinear 7 cm. Sample; whole antibody 3.2 μg, heavy and light chain: 1.6 μg. Staining; deep purple (GE Healthcare). (a) InfA-9 monoclonal antibody. Whole antibody: many spots are seen in the whole antibody of InfA-9. The clear four spots (pI = 6.0, 6.1, 6.2 and 6.5) in the heavy chain and three spots (pI = 5.9, 6.1 and 6.5) in the light chain were detected. Heavy chain (H): clear five spots (pI = 6.1, 6.3, 6.5, 6.7 and 6.9) were detected. The pI positions of the spots were a little bit different compared to the whole antibody. Light chain (L): clear six spots (pI = 5.7, 5.9, 6.0, 6.1, 6.2 and 6.7) were detected. The number of spots increased compared with those of the whole antibody. (b) InfA-15 monoclonal antibody. For the whole antibody and the heavy and light chain, similar results showing many spots at different pI for the same molecular sizes were observed, suggesting that the molecular heterogeneity of antibodies are generally occurring events.

were a little bit different compared to the whole antibody. For the case of the light chain, clear six spots (pI = 5.7, 5.9, 6.0, 6.1, 6.2 and 6.7) were detected. The number of spots increased compared with those of the whole antibody. These phenomena are not exceptional, but general. The mAb, InfA-15, exhibited similar results. **Figure 1b** shows 2D-gel electrophoresis using InfA-15 mAb. In the results of the whole antibody and the heavy chain and the light chain, the pattern of spots were a little different from those of InfA-9 mAb, but several different pI spots were observed in all cases. Namely, different pI spots are present at the same molecular size in any mAb. Note that structural diversity (molecular heterogeneity) should be existing even in the monoclonal antibody and the subunits, while they are a single protein. In our case, it is considered that the various electrical charges of the molecule may be one of the causes.

### **2.2. Recombinant monoclonal antibody Herceptin**

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 capillary isoelectric focusing and 2D-gel electrophoresis.

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

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 reaction of the deamidation and the heterogeneity is lost.
