**Table 1.**

*Masses and sequences of peptides found in a glycopeptide-enriched fraction from porcine IgG (wild-type) tryptic digestion products. Red: Labeled manually; black: Sequenced and/or matched by GPM [24]. Glycopeptides, m/z values in bold not observed, only glycoforms.*

**75**

**Figure 2.**

**followed by SEC**

*EEQFNSTYR, (d) Man-9 of undetermined peptide.*

*Characterization of Whole and Fragmented Wild-Type Porcine IgG*

**3.2 Fragmentation of porcine wild-type IgG with papain and fabulous™** 

*Tandem mass spectra of doubly or triply charged ions of glycopeptides enriched from the tryptic products of porcine wild-type IgG. (a) G0F glycoform of EAQFNSTYR, (b) G0F of undetermined peptide, (c) G1FS of* 

Antibody samples were first fragmented on immobilized papain, and thus it is expected that only IgG-related products will be present in the mixture. As shown by **Figure 3a**, all antibody was fragmented (intact antibody would have appeared at ca. 4.5 min). In general, when IgG is incubated with papain with a reducing agent, one or more peptide bonds in the hinge region are split, producing three fragments of similar size: two Fab fragments and one Fc fragment. The Fc may remain intact

*DOI: http://dx.doi.org/10.5772/intechopen.82792*

#### *Recent Advances in Analytical Chemistry*

**Figure 2.**

*Recent Advances in Analytical Chemistry*

**74**

**Peptide sequence**

Unknown TVIYSTNSRPTGVPSR

QLIYSTNNRPTGVPSR

QLIYQTNSRPTGVPSR

SSSGFTCQVTHEGTIVEK

AAPTVNLFPPSSEELGTNK

ASGVPDRFSGSGSGTDFTLK

FTDETLVSDLQPSLDRAR

ATLVCLISDFYPGAVTVWK

AGPLGWFERRPEPPPGPPSK

QSNNKYAASSYLALSASDWK

Unknown Unknown

*m/z* **(M + H)+**

1718.91 1734.91 1802.95 1816.97 1966.92

1972 1985.96 2063.04

2083.1 2172.14 2204.06 2717.07 2832.04 *P01857, human IgG heavy chain; IgGs. from [3]; IPR, GPM entries.*

**Table 1.**

*GPM [24]. Glycopeptides, m/z values in bold not observed, only glycoforms.*

*\*L8B180,L8B0Z4,L8B0S2,L8B0S7,K7ZLA7 porcine IgG heavy chain entries (Fc) [22]; P01846, porcine IgG lambda constant domain (Fab); K7ZJP7, IgM heavy chain; P01786, mouse IgG heavy chain;* 

*Masses and sequences of peptides found in a glycopeptide-enriched fraction from porcine IgG (wild-type) tryptic digestion products. Red: Labeled manually; black: Sequenced and/or matched by* 

1.73 3.88 1.98 1.32 1.06 1.86 0.15 3.26 2.39 0.82

**Error (ppm)**

*m/z* **glycoform**

**Glycoform**

**Identification source\***

Unavailable

P01846

Unavailable

IPR 007110 (Ig-like)

P01846

P01846

Unavailable

Unavailable

P01846

Unavailable

P01846

Unavailable

Unavailable

*Tandem mass spectra of doubly or triply charged ions of glycopeptides enriched from the tryptic products of porcine wild-type IgG. (a) G0F glycoform of EAQFNSTYR, (b) G0F of undetermined peptide, (c) G1FS of EEQFNSTYR, (d) Man-9 of undetermined peptide.*

#### **3.2 Fragmentation of porcine wild-type IgG with papain and fabulous™ followed by SEC**

Antibody samples were first fragmented on immobilized papain, and thus it is expected that only IgG-related products will be present in the mixture. As shown by **Figure 3a**, all antibody was fragmented (intact antibody would have appeared at ca. 4.5 min). In general, when IgG is incubated with papain with a reducing agent, one or more peptide bonds in the hinge region are split, producing three fragments of similar size: two Fab fragments and one Fc fragment. The Fc may remain intact

based on conditions and enzyme used [9, 10]. The cleavages occur at cysteines around position 271 (in **Figure A1**), about 10 amino acids from the IdeS cleavage site [29]. In human IgG subtypes (IgG1-4), there are on average of three cysteines in the range of hinge region positions 265–275 to make the numbers correspond with those of **Figure A1**, where papain cleavages can be initiated [29]. Porcine IgG has similar cysteine motifs in these positions; however, **Figure A1** shows different lengths of amino acid chains in the hinge region, which did not seem to prevent fragmentation. Interestingly in this papain-fragmentation experiment, the Fc did not remain intact, but the Fab did. **Figure 3a** shows significant separation although not at the baseline, but which still allowed the collection of Fab and Fc fractions.

In order to further isolate Fab and Fc segments, collected fractions were analyzed in turn by SDS-PAGE. This allowed identification by mass, with confirmation by linear mode MALDI-TOF-MS (not shown) and in-gel tryptic digestion of the bands (analyzed by reflector mode MALDI-TOF-MS and HPLC-MS, next section). The first major peak in **Figure 3a** was identified as originating from the Fab, and the second peak was identified as the Fc. The intact Fab fragment is larger and thus elutes prior to the split Fc, while the opposite is usually true in the case of human IgG [15].

Fabulous™ is a recombinant cysteine protease which under reducing conditions digests in the hinge region of antibodies from many species and subclasses, including human, mouse, rat, and rhesus monkey, yielding Fab and Fc fragments [11]. Some specific fragmentation site information is available for human IgG1, mouse IgG1, and rabbit IgG, but no information at all pertains to the fragmentation of porcine

#### **Figure 3.**

*Size exclusion chromatograms obtained for 200 μg of wild-type porcine IgG fragmented by (a) immobilized papain and (b) Fabulous™.*

**77**

**Figure 4.**

*Characterization of Whole and Fragmented Wild-Type Porcine IgG*

depicted in a and b were performed several months apart.

**3.3 SDS-PAGE separation of porcine IgG fragments**

papain's sequence [31].

IgG. Looking at all porcine IgG subtype sequences (see alignment in **Figure A1**), they have a motif similar to that of rabbit IgG (KP270I/CPP) [11], that is, with a potential fragmentation site between isoleucine I and cysteine C: CP270I/CPG or CP270I/CPA. In general, when IgG molecules are incubated with Fabulous™ in the presence of a reducing agent, one or more peptide bonds in the hinge region are split next to a cysteine, producing two Fab fragments and one Fc. As the reducing agent is present during the digestion reaction, it is likely that all interchain thiols will be reduced. In this experiment with Fabulous™ using 100 μg of pig IgG, a small portion of the antibody was not fragmented; otherwise the Fc was split into two halves and the Fab fragments remained intact. The HPLC-SEC chromatogram is shown in **Figure 3b**. The first major peak was indicative of the Fab fragment, and the second peak eluting after identified as the Fc, as verified later by SDS-PAGE and further tryptic digestion. As noticed in the chromatograms of **Figure 3**, elution times are different, that is, longer in portion b. This is due to gradual deterioration of the column, as both experiments

The fact that Fabulous™ (28,724 Da [11]) was free in solution and not immobilized as in the case of papain involves that it would elute in the SEC chromatogram (**Figure 3b**), most probably in the second peak with the Fc. If this proprietary enzyme has a sequence similar to that of papain, subsequent proteolysis by trypsin is likely to occur extensively, as many lysine and arginine residues are present in

For papain-produced fragments, a nonreducing gel experiment was performed on the previously collected SEC fractions (**Figure 4a**). This experiment allowed confirming the identity of the fragments. A reducing SDS-PAGE experiment was also performed on intact pig IgG to serve as a control (**Figure 4b**). Bands were then excised from the gel, followed by in-gel tryptic digestion. This helped to single out heavy and light chains for differentiating between Fab and Fc according to their respective known glycopeptides and peptides [2, 3]. In **Figure 4a**, lanes 6–9 correspond to a fraction containing both Fab and Fc collected at the junction of SEC peaks in **Figure 3a**. A single 50 kDa Fab band and two 25 kDa bands were obtained. The Fab band is positioned just below the 50 kDa marker band and clearly below the Fc band in **Figure 4b**, and the Fab should have a lower molecular weight than intact Fc [32].

*(a) Fab and Fc SEC fractions from papain-fragmented porcine IgG on nonreducing SDS-PAGE and (b) from* 

*Fabulous™-fragmented porcine IgG, (c) whole porcine IgG runs under reducing conditions.*

*DOI: http://dx.doi.org/10.5772/intechopen.82792*

#### *Characterization of Whole and Fragmented Wild-Type Porcine IgG DOI: http://dx.doi.org/10.5772/intechopen.82792*

*Recent Advances in Analytical Chemistry*

the opposite is usually true in the case of human IgG [15].

based on conditions and enzyme used [9, 10]. The cleavages occur at cysteines around position 271 (in **Figure A1**), about 10 amino acids from the IdeS cleavage site [29]. In human IgG subtypes (IgG1-4), there are on average of three cysteines in the range of hinge region positions 265–275 to make the numbers correspond with those of **Figure A1**, where papain cleavages can be initiated [29]. Porcine IgG has similar cysteine motifs in these positions; however, **Figure A1** shows different lengths of amino acid chains in the hinge region, which did not seem to prevent fragmentation. Interestingly in this papain-fragmentation experiment, the Fc did not remain intact, but the Fab did. **Figure 3a** shows significant separation although not at the baseline, but which still allowed the collection of Fab and Fc fractions. In order to further isolate Fab and Fc segments, collected fractions were analyzed in turn by SDS-PAGE. This allowed identification by mass, with confirmation by linear mode MALDI-TOF-MS (not shown) and in-gel tryptic digestion of the bands (analyzed by reflector mode MALDI-TOF-MS and HPLC-MS, next section). The first major peak in **Figure 3a** was identified as originating from the Fab, and the second peak was identified as the Fc. The intact Fab fragment is larger and thus elutes prior to the split Fc, while

Fabulous™ is a recombinant cysteine protease which under reducing conditions digests in the hinge region of antibodies from many species and subclasses, including human, mouse, rat, and rhesus monkey, yielding Fab and Fc fragments [11]. Some specific fragmentation site information is available for human IgG1, mouse IgG1, and rabbit IgG, but no information at all pertains to the fragmentation of porcine

*Size exclusion chromatograms obtained for 200 μg of wild-type porcine IgG fragmented by (a) immobilized* 

**76**

**Figure 3.**

*papain and (b) Fabulous™.*

IgG. Looking at all porcine IgG subtype sequences (see alignment in **Figure A1**), they have a motif similar to that of rabbit IgG (KP270I/CPP) [11], that is, with a potential fragmentation site between isoleucine I and cysteine C: CP270I/CPG or CP270I/CPA.

In general, when IgG molecules are incubated with Fabulous™ in the presence of a reducing agent, one or more peptide bonds in the hinge region are split next to a cysteine, producing two Fab fragments and one Fc. As the reducing agent is present during the digestion reaction, it is likely that all interchain thiols will be reduced.

In this experiment with Fabulous™ using 100 μg of pig IgG, a small portion of the antibody was not fragmented; otherwise the Fc was split into two halves and the Fab fragments remained intact. The HPLC-SEC chromatogram is shown in **Figure 3b**. The first major peak was indicative of the Fab fragment, and the second peak eluting after identified as the Fc, as verified later by SDS-PAGE and further tryptic digestion. As noticed in the chromatograms of **Figure 3**, elution times are different, that is, longer in portion b. This is due to gradual deterioration of the column, as both experiments depicted in a and b were performed several months apart.

The fact that Fabulous™ (28,724 Da [11]) was free in solution and not immobilized as in the case of papain involves that it would elute in the SEC chromatogram (**Figure 3b**), most probably in the second peak with the Fc. If this proprietary enzyme has a sequence similar to that of papain, subsequent proteolysis by trypsin is likely to occur extensively, as many lysine and arginine residues are present in papain's sequence [31].

#### **3.3 SDS-PAGE separation of porcine IgG fragments**

For papain-produced fragments, a nonreducing gel experiment was performed on the previously collected SEC fractions (**Figure 4a**). This experiment allowed confirming the identity of the fragments. A reducing SDS-PAGE experiment was also performed on intact pig IgG to serve as a control (**Figure 4b**). Bands were then excised from the gel, followed by in-gel tryptic digestion. This helped to single out heavy and light chains for differentiating between Fab and Fc according to their respective known glycopeptides and peptides [2, 3]. In **Figure 4a**, lanes 6–9 correspond to a fraction containing both Fab and Fc collected at the junction of SEC peaks in **Figure 3a**. A single 50 kDa Fab band and two 25 kDa bands were obtained. The Fab band is positioned just below the 50 kDa marker band and clearly below the Fc band in **Figure 4b**, and the Fab should have a lower molecular weight than intact Fc [32].

#### **Figure 4.**

*(a) Fab and Fc SEC fractions from papain-fragmented porcine IgG on nonreducing SDS-PAGE and (b) from Fabulous™-fragmented porcine IgG, (c) whole porcine IgG runs under reducing conditions.*

Both bands appearing in the Fc region were digested independently with trypsin as denoted by "upper Fc band" and "lower Fc band." It appears from further results that they belong to different IgG subtypes. For Fabulous™ fragments, the same experiment was conducted and results are shown in **Figure 4b**. According to gel separations, papain and Fabulous™ had very similar fragmentation effects on porcine IgG.

#### **3.4 MALDI-MS of in-gel tryptic-digested Fc bands**

**Figure 5a** shows the tryptic products for the upper Fc band (papain generated) in **Figure 4a**. The main glycopeptide observed has three main glycoforms at *m/z* 2472, 2618, and 2780 as [M + H]+ ions. In **Figure 5b** (lower Fc band), two series of glycopeptides are observed, that is, the same as above and another one at *m/z* 2414, 2560, and 2722. The amino acid sequences of these glycopeptides were verified by MALDI-MS/MS as EEQFNSTYR and EAQFNSTYR, respectively [2, 3], with glycoforms as indicated in the figure. PNGase de-glycosylation of these two samples led to the spectrum of **Figure 5c**, where peptides at *m/z* 1117 (EAQFDSTYR) and 1175 (EEQFDSTYR) were detected, while the analysis of released glycans was not successful. Similar MALDI-MS and MS/MS results were obtained for the Fabulous™ generated porcine IgG that combined upper and lower Fc fragments digested with trypsin. Besides glycopeptides from the Fc region, other Fc ɤ-domain peptides were observed, as identified from the database [22] in **Table 2**.

#### **Figure 5.**

*Reflector positive mode MALDI-TOF-MS spectra of (a) tryptic digestion products from pig IgG Fc fragment (higher 25 kDa band) and (b) lower Fc fragment. Both bands were excised from the gel run under nonreducing conditions shown in Figure 4a. (c) De-glycosylated peptides after removal of glycans to give peptides at m/z 1117 and 1175.*

**79**

**Peptide sequence**

Unknown Unknown Unknown DLPAPITR

Unknown Unknown Unknown Unknown Unknown EAQFNSTYR

Unknown EEQFNSTYR

Unknown Unknown VNNVDLPAPITR

Unknown SNGQPEPEGNYR

Unknown Unknown Unknown Unknown Unknown LVESGGGLVQPGGSLR

*m/z* **(M + H)+**

807.28 842.38 870.41

882.5 905.33 951.33 993.36 1033.4 1107.4 **1115.5** 1165.4 **1173.50**

1209.5 1261.4 1308.6 1330.4 1347.6 1374.4 1392.2 1427.6 1475.6 1503.4 1525.7

−74.23

−1.48

−66.51

−14.6

2471.60 2779.62 2779.63

G0 G0F G1F

−22.51

2560.39

G0F

−5.78

**Error ppm**

*m/z* **glyco-form**

**Glyco-form**

**Source\*** Unavailable Unavailable Unavailable IgG2ba-b,IgG4a-b,IgG6a-b, L8B180,L8B0S7,L8B0Z4

Unavailable Unavailable Unavailable Unavailable Unavailable IgG6a,L8B180,L8B0Z4

Unavailable

IgG1a-b,IgG2a-b,IgG4a-b,IgG5a-b,

IgG6b,L8B0S7,L8B0S2,K7ZLA7

Unavailable

Unavailable

IgG1a-b,K7ZLA7

Unavailable

IgG1a,IgG6a-b,L8B180,L8B0S2, K7ZLA7,L8B0Z4

Unavailable

Unavailable

Unavailable

Unavailable

Unavailable

L8B180,L8B0S7

*Characterization of Whole and Fragmented Wild-Type Porcine IgG*

*DOI: http://dx.doi.org/10.5772/intechopen.82792*


#### *Characterization of Whole and Fragmented Wild-Type Porcine IgG DOI: http://dx.doi.org/10.5772/intechopen.82792*

*Recent Advances in Analytical Chemistry*

2472, 2618, and 2780 as [M + H]+

**3.4 MALDI-MS of in-gel tryptic-digested Fc bands**

observed, as identified from the database [22] in **Table 2**.

Both bands appearing in the Fc region were digested independently with trypsin as denoted by "upper Fc band" and "lower Fc band." It appears from further results that they belong to different IgG subtypes. For Fabulous™ fragments, the same experiment was conducted and results are shown in **Figure 4b**. According to gel separations, papain and Fabulous™ had very similar fragmentation effects on porcine IgG.

**Figure 5a** shows the tryptic products for the upper Fc band (papain generated) in **Figure 4a**. The main glycopeptide observed has three main glycoforms at *m/z*

glycopeptides are observed, that is, the same as above and another one at *m/z* 2414, 2560, and 2722. The amino acid sequences of these glycopeptides were verified by MALDI-MS/MS as EEQFNSTYR and EAQFNSTYR, respectively [2, 3], with glycoforms as indicated in the figure. PNGase de-glycosylation of these two samples led to the spectrum of **Figure 5c**, where peptides at *m/z* 1117 (EAQFDSTYR) and 1175 (EEQFDSTYR) were detected, while the analysis of released glycans was not successful. Similar MALDI-MS and MS/MS results were obtained for the Fabulous™ generated porcine IgG that combined upper and lower Fc fragments digested with trypsin. Besides glycopeptides from the Fc region, other Fc ɤ-domain peptides were

*Reflector positive mode MALDI-TOF-MS spectra of (a) tryptic digestion products from pig IgG Fc fragment (higher 25 kDa band) and (b) lower Fc fragment. Both bands were excised from the gel run under nonreducing conditions shown in Figure 4a. (c) De-glycosylated peptides after removal of glycans to give peptides* 

ions. In **Figure 5b** (lower Fc band), two series of

**78**

**Figure 5.**

*at m/z 1117 and 1175.*


**Table 2.**

**81**

*Characterization of Whole and Fragmented Wild-Type Porcine IgG*

**3.5 In-gel tryptic-digested fab bands analyzed by MALDI-MS**

peptide masses and sequences available are listed in **Table 3**

FSGAISGNK 880.45 880.39

FSGSGSGTDFTLK 1303.62 1303.61

**Sequence** *m/z* **ESI** *m/z* **MALDI Error** 

The bands just below 50 kDa from both papain and Fabulous™ digestion processes were excised from the gel and digested with trypsin. MALDI-MS spectra of the products were obtained, and display no obvious glycopeptides as found in the Fc. Some peaks could be identified as light and heavy chain Fab peptides. Fab

Unknown 951.33 951.21 Unavailable Unknown 993.36 993.56 Unavailable Unknown 1052.22 Unavailable Unknown 1119.29 Unavailable Unknown 1141.26 Unavailable Unknown 1209.48 1209.28 Unavailable Unknown 1242.51 Unavailable

Unknown 1339.55 Unavailable Unknown 1374.27 Unavailable Unknown 1383.95 Unavailable Unknown 1419.30 Unavailable Unknown 1476.02 Unavailable Unknown 1503.39 1504.12 Unavailable Unknown 1526.39 Unavailable Unknown 1584.35 Unavailable Unknown 1622.45 Unavailable YAASSYLALSASDWK 1632.79 1632.85 36.7 P01846 (Ig

Unknown 1660.16 Unavailable Unknown 1735.19 Unavailable Unknown 1762.23 Unavailable QLIYSTNNRPTGVPSR 1802.95 1803.00 27.73 Unavailable Unknown 1826.41 Unavailable Unknown 1866.38 Unavailable Unknown 1910.24 Unavailable Unknown 1942.26 Unavailable AAPTVNLFPPSSEELGTNK 1972.00 1972.18 90.2 P01846 (Ig

Unknown 2034.37 Unavailable FTDETLVSDLQPSLDRAR 2063.04 2063.17 62.9 Unavailable Unknown 2135.81 2136.40 Unavailable Unknown 2210.95 2211.46 Unavailable Unknown 2338.64 Unavailable Unknown 2377.63 Unavailable Unknown 2408.38 Unavailable VTLTCLVTGFYPPDIDVEWQR 2509.24 2509.39 58.5 IgG4a

.

**ppm**

**Identification source**

const. region)

λ

λ

λ

const. region)

const. region)

−70.8 P01846 (Ig

−4.91 Unavailable

*DOI: http://dx.doi.org/10.5772/intechopen.82792*

*Masses and sequences of peptides found by MALDI-MS in porcine IgG (wild-type) tryptic digestion products of combined Fc bands obtained by papain and Fabulous™ fragmentation. Peptides* 

*and glycopeptides in red were sequenced manually after MS/MS and/or tentatively identified by mass fingerprinting.*

#### *Recent Advances in Analytical Chemistry*

*Recent Advances in Analytical Chemistry*

**80**

**Peptide sequence**

VSSQNIQDFPSVLR

YAASSYLALSASDWK

VVSVLPIQHQDWLK

Unknown Unknown Unknown Unknown Unknown STGKPTLYNVSLVLSDT

EPQVYTLSPSAEELSR

EPQVYTLPPPAEELSR

EPQVYTLPPPTEELSR

TTPPQQDVDGTFFLYSK

TTPPQQDVDGTYFLYSK

AAPTVNLFPPSSEELGTNK

Unknown Unknown Unknown GLEGLAYIGYTGVITDYADSVK

*P01857, human IgG heavy chain; IgGs, from [3]; IPR, GPM entries.*

**Table 2.**

*m/z* **(M + H)+**

1589.8 1632.8

1662 1677.5 1693.7 1725.4 1740.6 1754.4 1794.8 1805.8 1825.9 1855.9 1943.8 1959.9 1971.9 2135.8 2162.9

2211 2305.4

86.8 *\*L8B180,L8B0Z4,L8B0S2,L8B0S7,K7ZLA7, porcine IgG heavy chain entries (Fc) [22]; P01846, porcine IgG lambda constant domain (Fab); K7ZJP7, IgM heavy chain; P01786, mouse IgG heavy chain;* 

*Masses and sequences of peptides found by MALDI-MS in porcine IgG (wild-type) tryptic digestion products of combined Fc bands obtained by papain and Fabulous™ fragmentation. Peptides* 

*and glycopeptides in red were sequenced manually after MS/MS and/or tentatively identified by mass fingerprinting.*

−36.6

−1.07

−50.7

−2.32

2.9

−34.1

−60.4

13.7

−24.5

42.9

**Error ppm**

*m/z* **glyco-form**

**Glyco-form**

**Source\***

K7ZJP7 (IgM HC const. region)

P01846 UniprotKB (Ig λ const. region)

IgG1a-b,IgG3,IgG6b,L8B180,K7ZLA7, L8B0Z4

Unavailable

Unavailable

Unavailable

Unavailable

Unavailable

K7ZJP7 (IgM HC const. region)

IgG6a-b,L8B180,L8B0Z4

IgG1a-b,K7ZLA7

IgG4b

IgG1a-b,K7ZLA7

IgG2a-b,IgG3,IgG4a-b,IgG6a,L8B180, L8B0S7,L8B0Z4

P01846 UniprotKB (Ig λ const. region)

Unavailable

Unavailable

Unavailable

L8B180

#### **3.5 In-gel tryptic-digested fab bands analyzed by MALDI-MS**

The bands just below 50 kDa from both papain and Fabulous™ digestion processes were excised from the gel and digested with trypsin. MALDI-MS spectra of the products were obtained, and display no obvious glycopeptides as found in the Fc. Some peaks could be identified as light and heavy chain Fab peptides. Fab peptide masses and sequences available are listed in **Table 3**.



#### **Table 3.**

*Fab tryptic peptides from porcine IgG fragmented with papain and fabulous™. Second column (m/z ESI): Already identified in Table 1. Error: Measured using the MALDI m/z values against calculated values. Sequences in red are assigned from mass only based on Table 1.*

Overall, MALDI-TOF-MS experiments allowed suggesting that efficient separation of Fab and Fc fragments from each other occurred, using the SEC/SDS-PAGE procedure. There was a minimal number of overlapping peptides between Fab and Fc MALDI spectra (**Tables 2** and **3**). In **Table 3**, peptides in red had already been sequenced and appeared in **Table 1**; peptides whose *m/z* values appear in the second column were also observed in **Table 1**, although not sequenced. Overlapping peptides between **Tables 2** and **3** are at *m/z* 2509 (unknown), 2884 (glycoform of *m/z* 1115), and *m/z* 1677 (IgM). The presence of IgM in the sample had been noticed in an earlier report [3].

#### **3.6 Tryptic products of Fabulous™-fragmented porcine IgG analyzed by HPLC/MS**

Each sample of digestion products from Fc and Fab bands were analyzed twice by HPLC/MS, and results are summarized in **Table 4**. In **Figure A2**, total ion chromatograms (TIC) of the pig Fab and Fc tryptic products showed differences in the retention times of some peaks as expected. Peaks observed at the same retention times (e.g., 4.41–4.46, 10.35, and 23.10 min) and common to all injections corresponded to singly charged ions of small compounds of *m/z* ˂ 700 and were not considered in the analysis, as they probably were non-peptide contaminants. Peaks at 5.96 min also present in both chromatograms corresponded to a mass 1660 peptide from the Fab, AGGTTVTQVETTKPSK. Non-glycosylated peptides in **Table 4** were identified through MS/MS and database search, while glycopeptides were assigned by mass only in reference to entries in **Table 1**.

As Fabulous™ was possibly still present in the mixture at the time of tryptic digestion of the Fc gel band, analogous papain tryptic digestion products were sought for by *m/z*, but not found. Only one papain peptide with sequence YIDETNK (*m/z* 882.4203) could be present according to its nominal mass; however, the observed *m/z* 882.4998 peptide ions are closer to the calculated mass of IgG DLPAPITR, 882.4939. As seen in **Table 4**, many peptides remain un-sequenced/unidentified, and others are identified as either Fab or Fc peptides of porcine IgG. Interestingly, there were some very good matches with human and mouse IgG, which is not surprising given relatively high levels of homology between mammal species in general. However even with all the heavy chain sequences available in the literature for porcine IgG (see footnote of **Table 4** and **Figure A1**), none of them contained the human- and murine-assigned peptides. It is also interesting to observe that most peptides are predominant in either the Fab or Fc injections and that overlapping peptides are clearly more present in one sample than in the other. Accordingly, all Fab P01846 peptides (*m/z* 880, 1175, 1274, 1375, 1533, 1590, and 1661) were more abundant in the Fab than the Fc sample, although all these peptides showed some level of overlap. The same reasoning is applicable to Fc peptides, including the *N*-glycoforms of EAQFNSTYR and EEQFNSTYR. It is also possible to know the origin of most unidentified peptides in **Table 4** by comparing their normalized abundances. Other *N*-glycopeptides reported in **Table 1** were not detected in these Fab and Fc RPLC/MS experiments, possibly due to the lack of glycopeptide enrichment and to sample loss during the SEC and SDS-PAGE procedures.

**83**

**Mass (calc.)**

516.301 585.322 712.372 768.412 806.345 811.439 816.416 824.372 826.434

827.511 834.421 837.492 841.502 844.291 850.418 861.432 872.373 872.397 879.445 881.492

517.309 586.330 713.380 769.420 807.353 812.447 817.424 825.380 827.442 828.519 835.429 838.500 842.510 845.299 851.426 862.440 873.381 873.405 880.453 882.500

−0.45 −5.78

0.0

1559.7

479.8 410.6

FSGAISGNK

DLPAPITR

P01846

IgG2ba-b,IgG4a-b,

IgG6a-b,L8B180,L8B0S7,L8B0Z4

−5.12

14.2 977.9 264.0

0.0 220.3

0.0 0.0

52.3

74.7

15.5

42.4

0.0 1260.9

0.0

ALPAPIEK

P01857

−5.63

6.1 0.0 0.0 749.4 478.3

0.0

65.1

78.4

9.1 1176.6

166.0

0.00 −1.97 −4.25

8.0 0.0 652.0

76.1

0.0

*m/z* **(M + H) + (exp.)**

*m/z* **error (ppm)** **Pig Fab wt (avg)**

**Pig Fc wt (avg)**

23.9 1972.6 891.4

9.4 22.4 424.4

LVESGGGLV

VDKR PGGSLR LVESGGGL

IgG1a-b,IgG2a-b,IgG3,IgG4a-b

P01786 P01786

**Normalized abundance**

**Sequence**

**Sources of identification\***

*Characterization of Whole and Fragmented Wild-Type Porcine IgG*

P01786

*DOI: http://dx.doi.org/10.5772/intechopen.82792*


#### *Characterization of Whole and Fragmented Wild-Type Porcine IgG DOI: http://dx.doi.org/10.5772/intechopen.82792*

*Recent Advances in Analytical Chemistry*

*Sequences in red are assigned from mass only based on Table 1.*

**HPLC/MS**

**Table 3.**

Overall, MALDI-TOF-MS experiments allowed suggesting that efficient separation of Fab and Fc fragments from each other occurred, using the SEC/SDS-PAGE procedure. There was a minimal number of overlapping peptides between Fab and Fc MALDI spectra (**Tables 2** and **3**). In **Table 3**, peptides in red had already been sequenced and appeared in **Table 1**; peptides whose *m/z* values appear in the second column were also observed in **Table 1**, although not sequenced. Overlapping peptides between **Tables 2** and **3** are at *m/z* 2509 (unknown), 2884 (glycoform of *m/z* 1115), and *m/z* 1677 (IgM).

Unknown 2691.50 Unavailable Unknown 2807.49 Unavailable

*Fab tryptic peptides from porcine IgG fragmented with papain and fabulous™. Second column (m/z ESI): Already identified in Table 1. Error: Measured using the MALDI m/z values against calculated values.* 

**ppm**

**Identification source**

The presence of IgM in the sample had been noticed in an earlier report [3].

**Sequence** *m/z* **ESI** *m/z* **MALDI Error** 

**3.6 Tryptic products of Fabulous™-fragmented porcine IgG analyzed by** 

by HPLC/MS, and results are summarized in **Table 4**. In **Figure A2**, total ion chromatograms (TIC) of the pig Fab and Fc tryptic products showed differences in the retention times of some peaks as expected. Peaks observed at the same retention times (e.g., 4.41–4.46, 10.35, and 23.10 min) and common to all injections corresponded to singly charged ions of small compounds of *m/z* ˂ 700 and were not considered in the analysis, as they probably were non-peptide contaminants. Peaks at 5.96 min also present in both chromatograms corresponded to a mass 1660 peptide from the Fab, AGGTTVTQVETTKPSK. Non-glycosylated peptides in **Table 4** were identified through MS/MS and database search, while glycopeptides

were assigned by mass only in reference to entries in **Table 1**.

enrichment and to sample loss during the SEC and SDS-PAGE procedures.

Each sample of digestion products from Fc and Fab bands were analyzed twice

As Fabulous™ was possibly still present in the mixture at the time of tryptic digestion of the Fc gel band, analogous papain tryptic digestion products were sought for by *m/z*, but not found. Only one papain peptide with sequence YIDETNK (*m/z* 882.4203) could be present according to its nominal mass; however, the observed *m/z* 882.4998 peptide ions are closer to the calculated mass of IgG DLPAPITR, 882.4939. As seen in **Table 4**, many peptides remain un-sequenced/unidentified, and others are identified as either Fab or Fc peptides of porcine IgG. Interestingly, there were some very good matches with human and mouse IgG, which is not surprising given relatively high levels of homology between mammal species in general. However even with all the heavy chain sequences available in the literature for porcine IgG (see footnote of **Table 4** and **Figure A1**), none of them contained the human- and murine-assigned peptides. It is also interesting to observe that most peptides are predominant in either the Fab or Fc injections and that overlapping peptides are clearly more present in one sample than in the other. Accordingly, all Fab P01846 peptides (*m/z* 880, 1175, 1274, 1375, 1533, 1590, and 1661) were more abundant in the Fab than the Fc sample, although all these peptides showed some level of overlap. The same reasoning is applicable to Fc peptides, including the *N*-glycoforms of EAQFNSTYR and EEQFNSTYR. It is also possible to know the origin of most unidentified peptides in **Table 4** by comparing their normalized abundances. Other *N*-glycopeptides reported in **Table 1** were not detected in these Fab and Fc RPLC/MS experiments, possibly due to the lack of glycopeptide

**82**


**85**

**Mass (calc.)**

1307.718 1308.702 1312.672 1316.625 1329.687 1330.670 1334.648 1340.548 1345.666 1346.587 1347.568 1350.620 1368.616 1374.732 1376.558 1390.608

1391.593 1399.808 1433.682 1449.688

1434.690 1450.696

1400.816

1392.601

1391.616

1377.566

1375.740

−2.30

742.1 457.8

3.4 6.6 1434.9

8.5 17.8

958.3

226.8

1503.7

898.0

308.0

61.0

219.7

TVTQGVETTKPSK

P01846

1369.624

1351.628

1348.576

1347.595

1.41

1346.674

1341.556

1335.656

1331.678

1330.695

1317.633

1309.710 1313.680

−1.22

1308.726

−1.30

*m/z* **(M + H) + (exp.)**

*m/z* **error (ppm)** **Pig Fab wt (avg)**

139.6

13.2 2.4 896.9 20.3 87.9 23.1 131.8

5.8 47.2 50.8

7.9 16.4

1702.0

512.8

484.9

**Pig Fc wt (avg)**

13772.8

3997.2 306.7 223.7 434.9 1324.0

374.1 45.0 2149.3 658.2

SNGQPEPEGNYR

IgG1a,IgG5b,IgG6b,L8B180K7ZLA7,L8

B0Z4

ESGGGLVQPGGSLR

P01786

VNNVDLPAPITR

IgG1a-b,K7ZLA7

**Normalized abundance**

**Sequence**

**Sources of identification\***

*Characterization of Whole and Fragmented Wild-Type Porcine IgG*

*DOI: http://dx.doi.org/10.5772/intechopen.82792*

#### *Recent Advances in Analytical Chemistry*


#### *Characterization of Whole and Fragmented Wild-Type Porcine IgG DOI: http://dx.doi.org/10.5772/intechopen.82792*

*Recent Advances in Analytical Chemistry*

**84**

**Mass (calc.)**

888.366

917.391 919.450 935.856 939.510 990.341 994.506 994.512 1043.899 1044.554

1057.831 1066.539 1103.601 1164.577 1174.613 1208.640 1238.648

1259.553 1260.536 1273.685 1296.678 1298.485 1302.609

1303.617

0.00

1299.493

1297.686

889.374 918.399 920.458 936.864 940.518 991.349 995.513 995.520 1044.907 1045.562 1058.839 1067.547 1104.609 1165.585 1175.621 1209.648 1239.656 1260.561 1261.544 1274.693

−1.89

−5.01

−1.47

−2.30

7.61

*m/z* **(M + H) + (exp.)**

*m/z* **error** 

**Normalized abundance**

**Sequence**

**Sources of identification\***

**(ppm)**

**Pig Fab wt (avg)**

0.0 254.9

17.9 2502.6

86.4 264.4

13.2

7.6 1589.5 5106.9 1179.7

0.0 0.0 66.4 468.4

0.0 1.6 3.7 15.5 324.0

9.3 30.8 2446.2

**Pig Fc wt (avg)**

44.9

54.6

135.1

2690.0

407.8

5.6

518.7

311.7

1690.0

6511.7 1190.5

8.5 17.0

199.4

169.5 790.8

343.5

480.5

1386.4

104.5 727.0

263.2

908.9

FSGSGSGTDFTLK

Unavailable

VTQGVETTKPSK

P01846

TQGVETTKPSK

P01846

VDGVEVHNAK

P01857

LSSPATLNSR

Unavailable

LVESGGGLVQ

P01786


**87**

**Mass (calc.)**

1801.950

1807.752 1824.931 1854.932 1862.867 1919.930 1958.924 2092.889

2251.915 2267.910 2289.908 2355.931 2413.960 2435.939 2451.911 2558.988 2576.008

2617.036

2093.897 2252.923 2268.918 2290.916 2356.939 2414.968 2436.947 2452.919 2559.995 2577.016 2618.044

−2.66

3.5

−19.69

0.0 0.0

−11.42

0.0 0.0 0.0

2793.9

86.5

90.0

25.6 439.0

1257.9

G0F EEQFNSTYR

IgG1a-b,IgG2a-b,IgG4a-b,IgG5ab,IgG6b,L8B0S7,L8B0S2,K7ZLA7

G0F EAQFNSTYR

IgG6a,L8B180,L8B0Z4

G0 of EAQFNSTYR

IgG6a,L8B180,L8B0Z4

1802.958 1808.760 1825.939 1855.940 1863.875 1920.938 1959.932

−1.07

3.4 649.7

0.0 0.0 0.0 0.0

14.8

750.1

356.8

72.8

62.0

2082.2

TTPPQQDVDGTYFLYSK

IgG4a-b, IgG6a, L8B180, L8B0S7, IgG2a, IgG2b, IgG3

−2.32

0.0 0.0 1.8

0.0

695.8

1499.6

2.90

4.0

670.0

EPQVYTLPPPAEELSR

EPQVYTLPPPTEELSR

3.60

191.0

4.8

287.3

*m/z* **(M + H) + (exp.)**

*m/z* **error (ppm)** **Pig Fab wt (avg)**

**Pig Fc wt (avg)**

99.2

QLIYSTNNRPTGVPSR

Unavailable

**Normalized abundance**

**Sequence**

**Sources of identification\***

*Characterization of Whole and Fragmented Wild-Type Porcine IgG*

*DOI: http://dx.doi.org/10.5772/intechopen.82792*

IgG1a-b,K7ZLA7

IgG4b

#### *Recent Advances in Analytical Chemistry*


#### *Characterization of Whole and Fragmented Wild-Type Porcine IgG DOI: http://dx.doi.org/10.5772/intechopen.82792*

*Recent Advances in Analytical Chemistry*

**86**

**Mass (calc.)**

1514.667 1524.822 1530.664

1532.803 1546.798 1562.772 1578.744 1589.823 1624.841 1642.851 1660.864

1682.829 1698.811 1719.703 1735.702 1753.805 1769.797

1590.831 1625.849 1643.859 1661.872 1683.837 1699.819 1720.711 1736.710 1754.813 1770.805

0.26

11688.6

346.8 1146.9

10.5 0.0 2.7 4.8

723.1

493.2

480.4

471.9

308.5

77.9

4028.5

−0.92

1827.2

614.7

AGGTTVTQGVETTKPSK

AGGTTVTQGVETTKPSK

[C-term] neutral loss

P01846

−1.93

647.8 321.8

72.8

181.7

GGTTVTQGVETTKPSK

P01846

1515.675 1525.830 1531.672 1533.811 1547.806 1563.780 1579.752

47.70

4.7

439.3

EEQFNSTYR +2GlcNAc

IgG1a-b,IgG2a-b,IgG4a-b,IgG5ab,IgG6b,L8B0S7,L8B0S2,K7ZLA7

−0.91

646.1

64.1 192.8

4750.7

2405.4

171.1

GTTVTQGVETTKPSK

P01846

−2.62

1005.8

0.3

599.0

20216.6

LVESGGGLVQPGGSLR

L8B180, L8B0S7

*m/z* **(M + H) + (exp.)**

*m/z* **error** 

**Normalized abundance**

**Sequence**

**Sources of identification\***

**(ppm)**

**Pig Fab wt (avg)**

100.8

**Pig Fc wt (avg)**

1436.0


*P01857, human IgG heavy chain; IgGs, from [3]; IPR, GPM entries.*

## **Table 4.**

**89**

**Acknowledgements**

Fabulous™ enzyme.

*Characterization of Whole and Fragmented Wild-Type Porcine IgG*

The initial RPLC-MS/MS experiment on the enriched whole IgG digest produced the most useful information in terms of glycopeptides. Besides the latter, many peptides were identified as originating from the Fc or Fab portion, while for other peptides origin remained unknown. This first workflow allowed

sequences could not be completely assigned. The second workflow necessitated more intensive work than the first one. Size-exclusion chromatography or SDS-PAGE was not sufficient on its own to properly separate Fab and Fc, and a combination of both was preferable. Even then, RPLC/MS results show partial overlaps for many peptides, although most peptides were much more abundant in one sample (Fc or Fab) than in the other. In terms of ionization modes used in the second workflow, ESI yielded more details on sialylated glycopeptides, but

were the result of depositing all tryptic products of a gel band onto one target spot, it is probable that the signals of sialylated glycopeptides or low concentra

tion Fab glycopeptides were overtaken competitively by peptides that are more

Porcine immunoglobulins constitute a complex ensemble of biomolecules, with several subtypes whose amino acid sequences are not clearly assigned and described in the literature (see **Figure A1**). In this study, three state-of-the-art mass spectrom

eters were used to characterize tryptic peptides, offering a considerable amount of complementary information owing to the great sensitivities of these instruments, given the small amounts of IgGs used. Indeed, fragmentation of porcine IgG into its Fc and Fab portions was achieved for the first time using papain and Fabulous™ on

μg or less of antibody. Fragments needed separation by both SEC and SDS-PAGE before analysis by MALDI-TOF-MS and HPLC/MS. These separation pro

cedures did not eliminate the overlapping of Fab and Fc tryptic peptides entirely; however, there was a significant level of discrimination. This workflow resulted in

During the course of this work, the several sources of sequence information on porcine IgG found in **Figure A1** were used to verify HPLC-MS/MS, MALDI-TOF-MS, and HPLC-MS data. Most Fc peptides were from the gamma constant region, where some Fab peptides were identified as belonging to the constant portion of the lambda chain. The HPLC-MS/MS method of tryptic peptide with

out previous Fab-Fc fragmentation was the most efficient in terms of useful data generated per amount of sample used, although many peptides could not be related to either Fc or Fab. This study highlights the need for detailed pertinent sequence information for porcine IgG, which is not a commonly studied set of biomolecules. Future work will involve the quantification of IgG subtypes according to unique

The authors thank Dr. Jean-Paul Soulillou and his team at the Université de Nantes, France, for providing pig IgG samples. Thanks to Genovis for donating

better knowledge about the origin of tryptic peptides from IgG.

peptide sequences that are already known in each subtype.

*N*-glycopeptides from the Fab portion of the antibody, although their

*N*-glycopeptides. As analyses by MALDI





*DOI: http://dx.doi.org/10.5772/intechopen.82792*

did not allow the observation of Fab

detecting

abundant.

200

**4. Conclusions**

**3.7 Comparative aspects of both workflows**

*Tryptic peptides from pig IgG Fab and Fc fragment analyzed by HPLC-MS/MS.*

#### **3.7 Comparative aspects of both workflows**

The initial RPLC-MS/MS experiment on the enriched whole IgG digest produced the most useful information in terms of glycopeptides. Besides the latter, many peptides were identified as originating from the Fc or Fab portion, while for other peptides origin remained unknown. This first workflow allowed detecting *N*-glycopeptides from the Fab portion of the antibody, although their sequences could not be completely assigned. The second workflow necessitated more intensive work than the first one. Size-exclusion chromatography or SDS-PAGE was not sufficient on its own to properly separate Fab and Fc, and a combination of both was preferable. Even then, RPLC/MS results show partial overlaps for many peptides, although most peptides were much more abundant in one sample (Fc or Fab) than in the other. In terms of ionization modes used in the second workflow, ESI yielded more details on sialylated glycopeptides, but did not allow the observation of Fab *N*-glycopeptides. As analyses by MALDI were the result of depositing all tryptic products of a gel band onto one target spot, it is probable that the signals of sialylated glycopeptides or low concentration Fab glycopeptides were overtaken competitively by peptides that are more abundant.

#### **4. Conclusions**

*Recent Advances in Analytical Chemistry*

**88**

**Mass (calc.)**

2639.006 2654.984 2670.955 2692.958 2779.092 2801.070

2817.033 2941.166 3004.216 3086.182

3005.224 3087.190 *P01857, human IgG heavy chain; IgGs, from [3]; IPR, GPM entries.*

**Table 4.**

*Tryptic peptides from pig IgG Fab and Fc fragment analyzed by HPLC-MS/MS.*

−1.33

0.0

2802.078 2818.041 2942.174

5.90

0.0 0.0

68.7

159.4 *\*L8B180,L8B0Z4,L8B0S2,L8B0S7,K7ZLA7, porcine IgG heavy chain entries (Fc) [22]; P01846, porcine IgG lambda constant domain (Fab); K7ZJP7, IgM heavy chain; P01786, mouse IgG heavy chain;* 

G1FS EEQFNSTYR

IgG1a-b,IgG2a-b,IgG4a-b,IgG5ab,IgG6b,L8B0S7,L8B0S2,K7ZLA7

2640.014 2655.992 2671.963 2693.966 2780.100

−1.36

0.0 2.8 0.0

244.1

380.8

266.4

G2F EEQFNSTYR

IgG1a-b,IgG2a-b,IgG4a-b,IgG5ab,IgG6b,L8B0S7,L8B0S2,K7ZLA7

*m/z* **(M + H) + (exp.)**

*m/z* **error** 

**Normalized abundance**

**Sequence**

**Sources of identification\***

**(ppm)**

**Pig Fab wt (avg)**

0.0 2.5 0.0 0.0

**Pig Fc wt (avg)**

706.3

1048.1

200.5

49.2

1327.0

G1F EEQFNSTYR

IgG1a-b,IgG2a-b,IgG4a-b,IgG5ab,IgG6b,L8B0S7,L8B0S2,K7ZLA7

> Porcine immunoglobulins constitute a complex ensemble of biomolecules, with several subtypes whose amino acid sequences are not clearly assigned and described in the literature (see **Figure A1**). In this study, three state-of-the-art mass spectrometers were used to characterize tryptic peptides, offering a considerable amount of complementary information owing to the great sensitivities of these instruments, given the small amounts of IgGs used. Indeed, fragmentation of porcine IgG into its Fc and Fab portions was achieved for the first time using papain and Fabulous™ on 200 μg or less of antibody. Fragments needed separation by both SEC and SDS-PAGE before analysis by MALDI-TOF-MS and HPLC/MS. These separation procedures did not eliminate the overlapping of Fab and Fc tryptic peptides entirely; however, there was a significant level of discrimination. This workflow resulted in better knowledge about the origin of tryptic peptides from IgG.

> During the course of this work, the several sources of sequence information on porcine IgG found in **Figure A1** were used to verify HPLC-MS/MS, MALDI-TOF-MS, and HPLC-MS data. Most Fc peptides were from the gamma constant region, where some Fab peptides were identified as belonging to the constant portion of the lambda chain. The HPLC-MS/MS method of tryptic peptide without previous Fab-Fc fragmentation was the most efficient in terms of useful data generated per amount of sample used, although many peptides could not be related to either Fc or Fab. This study highlights the need for detailed pertinent sequence information for porcine IgG, which is not a commonly studied set of biomolecules. Future work will involve the quantification of IgG subtypes according to unique peptide sequences that are already known in each subtype.

#### **Acknowledgements**

The authors thank Dr. Jean-Paul Soulillou and his team at the Université de Nantes, France, for providing pig IgG samples. Thanks to Genovis for donating Fabulous™ enzyme.

#### **Conflict of interest**

None of the authors has a conflict of interest.

#### **A.Appendix: supplementary materials**


**Figure A1.**

*Alignment of sequences available in the literature for the porcine IgG heavy chains. IgGn: from Ref. [3]. Others: UniprotKB [22].*

**91**

**Author details**

**Figure A2.**

Claudia Nelson1

Haley Neustaeter3

and Hélène Perreault1

*fragments, (a) Fc and (b) Fab.*

provided the original work is properly cited.

, Raymond Bacala<sup>2</sup>

\*

, Emy Komatsu1

2 Chemistry Department, University of Manitoba and

University of Manitoba, Winnipeg, MB, Canada

3 Chemistry Department and Department of Internal Medicine,

\*Address all correspondence to: helene.perreault@umanitoba.ca

*Characterization of Whole and Fragmented Wild-Type Porcine IgG*

*DOI: http://dx.doi.org/10.5772/intechopen.82792*

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Baylie Gigolyk1

, Oleg Krokhin3

*HPLC/MS total ion chromatograms obtained for the tryptic products of wild-type porcine IgG Fabulous™* 

1 Chemistry Department, University of Manitoba, Winnipeg, MB, Canada

Canadian Grain Commission, Government of Canada, Winnipeg, MB, Canada

4 Canadian Grain Commission, Government of Canada, Winnipeg, MB, Canada

, Evelyn Ang3

, Dave Hatcher4

,

*Characterization of Whole and Fragmented Wild-Type Porcine IgG DOI: http://dx.doi.org/10.5772/intechopen.82792*

#### **Figure A2.**

*Recent Advances in Analytical Chemistry*

None of the authors has a conflict of interest.

**A.Appendix: supplementary materials**

**Conflict of interest**

**90**

**Figure A1.**

*UniprotKB [22].*

*Alignment of sequences available in the literature for the porcine IgG heavy chains. IgGn: from Ref. [3]. Others:* 

*HPLC/MS total ion chromatograms obtained for the tryptic products of wild-type porcine IgG Fabulous™ fragments, (a) Fc and (b) Fab.*

#### **Author details**

Claudia Nelson1 , Raymond Bacala<sup>2</sup> , Baylie Gigolyk1 , Evelyn Ang3 , Haley Neustaeter3 , Emy Komatsu1 , Oleg Krokhin3 , Dave Hatcher4 and Hélène Perreault1 \*

1 Chemistry Department, University of Manitoba, Winnipeg, MB, Canada

2 Chemistry Department, University of Manitoba and Canadian Grain Commission, Government of Canada, Winnipeg, MB, Canada

3 Chemistry Department and Department of Internal Medicine, University of Manitoba, Winnipeg, MB, Canada

4 Canadian Grain Commission, Government of Canada, Winnipeg, MB, Canada

\*Address all correspondence to: helene.perreault@umanitoba.ca

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 3

Separation Approaches

and Validation

## Section 3
