6. Method transfer

With the advancement of the liquid chromatographic instruments from HPLC to UHPLC and with the advancement in column packing technology with smaller particle sizes, an easy method transfer from HPLC to UHPLC is becoming necessary. UHPLC has much lower extra column volume and can withstand more than 1000 bar besides other optimized instrumental features while conventional HPLC has maximum operable pressure of 400 bars. Differences between HPLC and UHPLC instruments give rise to many challenges when a method needs to be transferred from one system to another and if equivalent separation profiles are obtained. On the other hand, there are analysts who do not possess the UHPLC but still want to get advantage of smaller particle size SEC columns while using a traditional HPLC instrument. The effect of extra column volume in band broadening in conventional HPLC needs to be reduced and optimized by reducing the diffusion in the tubing between the injection valve and the column and between the column and the UV cell by using smaller ID tubing and micro flow cell. A column compatible with both UHPLC and conventional HPLC instruments may be helpful for easy method transfer in both cases.

As an example, an SEC HPLC method for the separation of a mAb using 5 μm, 7.8 mm ID 30 cm SEC column (TSKgel G3000SWXL) was transferred to a 2 μm, 4.6 mm ID 30 cm SEC column (TSKgel UP-SW3000 SEC) on a UHPLC instrument (Figure 30). The mobile phase and other chromatographic parameters were not changed except the flow rate reduced for the 2-μm column.

TSKgel UP-SW3000 columns feature the same pore size as the well-established TSKgel G3000SWxl columns. With the use of the 2-μm column, the resolution between peaks 2–3, 3– 4, 4–5, 5–6, and 6–7 increased respectively by 15, 25, 33, 24, and 37% compared to the 5-μm column. Retention time consistency was maintained, and similar separation profile was obtained at lower flow rate. A smaller 2-μm particle size column yielded a twofold higher sensitivity. The 2-μm columns yielded a back pressure acceptable for use in both HPLC and

Figure 31. Comparison of the separation efficiency of a 2-μm TSKgel UP-SW3000 column applied in HPLC and UHPLC mode. Conditions were the same as in Figure 30. The same standard protein mixture as in Figure 30 was used.

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Similarly, a method can be transferred directly from HPLC to UHPLC, without any change in

Desalting is a process to remove or reduce salt from the liquid, such as protein sample solution. Desalting by gel filtration chromatography (GFC) is the preferred method in biochemical laboratories to reduce the salt concentration or to exchange the buffer of a biopolymer solution. The main advantage of desalting by GFC over dialysis is the faster analysis time. Desalting may be needed for various reasons. Proteins eluting at high or elevated salt concentrations may need to be desalted to lower salt concentration prior to its use for the next step. Protein samples may also contain denaturants such as sodium dodecyl sulfate (SDS), guanidine hydrochloride, and urea which need to be removed. Desalting and buffer exchange of proteins or polynucleotides can also be performed by dialysis, ultra-filtration, or by using spin

conditions using 2 μm TSKgel UP-SW3000 columns as shown in Figure 31 [42].

UHPLC.

7. SEC for desalting


Figure 30. Comparison of the SEC HPLC method with the SEC UHPLC system. Separation efficiency of column TSKgel G3000SWXL (A) and column TSKgel UP-SW3000 SEC (B) was compared by loading a mixture of standard proteins (Thy = thyroglobulin bovine, γ-glo = γ-globulins from bovine blood, ova = albumin chicken egg grade VI, riboA = ribonuclease A type I-A from bovine pancreas, pAba = p-aminobenzoid acid) on both columns.

Separation of Monoclonal Antibodies by Analytical Size Exclusion Chromatography http://dx.doi.org/10.5772/intechopen.73321 165

Figure 31. Comparison of the separation efficiency of a 2-μm TSKgel UP-SW3000 column applied in HPLC and UHPLC mode. Conditions were the same as in Figure 30. The same standard protein mixture as in Figure 30 was used.

TSKgel UP-SW3000 columns feature the same pore size as the well-established TSKgel G3000SWxl columns. With the use of the 2-μm column, the resolution between peaks 2–3, 3– 4, 4–5, 5–6, and 6–7 increased respectively by 15, 25, 33, 24, and 37% compared to the 5-μm column. Retention time consistency was maintained, and similar separation profile was obtained at lower flow rate. A smaller 2-μm particle size column yielded a twofold higher sensitivity. The 2-μm columns yielded a back pressure acceptable for use in both HPLC and UHPLC.

Similarly, a method can be transferred directly from HPLC to UHPLC, without any change in conditions using 2 μm TSKgel UP-SW3000 columns as shown in Figure 31 [42].

#### 7. SEC for desalting

6. Method transfer

164 Antibody Engineering

2-μm column.

be helpful for easy method transfer in both cases.

With the advancement of the liquid chromatographic instruments from HPLC to UHPLC and with the advancement in column packing technology with smaller particle sizes, an easy method transfer from HPLC to UHPLC is becoming necessary. UHPLC has much lower extra column volume and can withstand more than 1000 bar besides other optimized instrumental features while conventional HPLC has maximum operable pressure of 400 bars. Differences between HPLC and UHPLC instruments give rise to many challenges when a method needs to be transferred from one system to another and if equivalent separation profiles are obtained. On the other hand, there are analysts who do not possess the UHPLC but still want to get advantage of smaller particle size SEC columns while using a traditional HPLC instrument. The effect of extra column volume in band broadening in conventional HPLC needs to be reduced and optimized by reducing the diffusion in the tubing between the injection valve and the column and between the column and the UV cell by using smaller ID tubing and micro flow cell. A column compatible with both UHPLC and conventional HPLC instruments may

As an example, an SEC HPLC method for the separation of a mAb using 5 μm, 7.8 mm ID 30 cm SEC column (TSKgel G3000SWXL) was transferred to a 2 μm, 4.6 mm ID 30 cm SEC column (TSKgel UP-SW3000 SEC) on a UHPLC instrument (Figure 30). The mobile phase and other chromatographic parameters were not changed except the flow rate reduced for the

Figure 30. Comparison of the SEC HPLC method with the SEC UHPLC system. Separation efficiency of column TSKgel G3000SWXL (A) and column TSKgel UP-SW3000 SEC (B) was compared by loading a mixture of standard proteins (Thy = thyroglobulin bovine, γ-glo = γ-globulins from bovine blood, ova = albumin chicken egg grade VI, riboA = ribonu-

clease A type I-A from bovine pancreas, pAba = p-aminobenzoid acid) on both columns.

Desalting is a process to remove or reduce salt from the liquid, such as protein sample solution. Desalting by gel filtration chromatography (GFC) is the preferred method in biochemical laboratories to reduce the salt concentration or to exchange the buffer of a biopolymer solution. The main advantage of desalting by GFC over dialysis is the faster analysis time. Desalting may be needed for various reasons. Proteins eluting at high or elevated salt concentrations may need to be desalted to lower salt concentration prior to its use for the next step. Protein samples may also contain denaturants such as sodium dodecyl sulfate (SDS), guanidine hydrochloride, and urea which need to be removed. Desalting and buffer exchange of proteins or polynucleotides can also be performed by dialysis, ultra-filtration, or by using spin columns. Desalting columns are characterized by a low exclusion limit and a large pore volume. Salts can fully access all pores, while proteins and other high MW species are excluded. Analytical columns packed with conventional packing materials such as dextran, cellulose, and polyacrylamide have limited physical stability and are not suitable when fast desalting is desired. Requirements for a fast desalting SEC column are [1] an inert matrix, [2] a large pore volume that is fully accessible to common salts and buffer components, [3] a pore size distribution that excludes the component(s) of interest from accessing the pores, and [4] sufficient mechanical strength to allow the use of the column in standard HPLC equipment. As an example, a 15-μm particle size TSKgel BioAssist DS column is composed of a stationary phase where the mechanical strength of the polyacrylamide gel is fourfold higher as compared to conventional gel by urea cross-linking. Conventionally, polyacrylamide beads have been prepared by reversed-phase suspension polymerization or by using a spray dry method. The uniform and more pressure-stable polyacrylamide beads packed in TSKgel BioAssist DS columns were prepared using a normal phase suspension method as shown in Figure 32 [43].

Fast desalting with excellent reproducibility could be carried out within 5 min using conventional HPLC system and TSKgel BioAssist DS Columns (4.6 mm ID and 10 mm ID) (Figure 33). All the proteins (see table below) eluted with the same retention time closer to void volume irrespective of their size (see the figures below), while salt and other small impurities eluted at longer retention time as a function of their size. Refractive index was used as a detector in this study since salts do not have any chromophore.

SEC columns designed for desalting using a HPLC instrument can be useful for the desalting of proteins and polynucleotides at analytical and semi-preparative scale.

8. Size exclusion chromatography column in the HILIC mode

stable polyacrylamide beads. The retention times of standard proteins and salt are shown.

applied for the separation of polar hydrophilic compounds.

very important.

mode.

For many years, SEC columns have been used to separate various nucleic acid species such as DNA, RNA, and tRNA as well as their constituent bases, adenine, guanine, thymine, cytosine, and uracil. In medicine, several primary nucleobases are the basis for the nucleoside analogues and other synthetic analogs which are used as anticancer and antiviral agents. Nucleobase modifications are the basis of oligonucleotide-based therapeutics, making their purification

Figure 33. Desalting of proteins using a TSKgel BioAssist DS column with 4.6 mm ID or 10 mm ID packed with pressure-

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Hydrophilic interaction chromatography (HILIC) is a variant of normal phase liquid chromatography which uses hydrophilic stationary phases with reversed-phase type eluents. It is

Chemically bonded diol-coated phases in size exclusion chromatography (SEC) columns demonstrate high polarity and hydrogen bonding properties. They do not contain ionizable groups compared to the unreacted free residual silanols, making them appropriate for the HILIC

Figure 32. Principle of the generation of pressure-stable polyacrylamide beads which can be packed in TSKgel BioAssist DS columns.

Separation of Monoclonal Antibodies by Analytical Size Exclusion Chromatography http://dx.doi.org/10.5772/intechopen.73321 167

columns. Desalting columns are characterized by a low exclusion limit and a large pore volume. Salts can fully access all pores, while proteins and other high MW species are excluded. Analytical columns packed with conventional packing materials such as dextran, cellulose, and polyacrylamide have limited physical stability and are not suitable when fast desalting is desired. Requirements for a fast desalting SEC column are [1] an inert matrix, [2] a large pore volume that is fully accessible to common salts and buffer components, [3] a pore size distribution that excludes the component(s) of interest from accessing the pores, and [4] sufficient mechanical strength to allow the use of the column in standard HPLC equipment. As an example, a 15-μm particle size TSKgel BioAssist DS column is composed of a stationary phase where the mechanical strength of the polyacrylamide gel is fourfold higher as compared to conventional gel by urea cross-linking. Conventionally, polyacrylamide beads have been prepared by reversed-phase suspension polymerization or by using a spray dry method. The uniform and more pressure-stable polyacrylamide beads packed in TSKgel BioAssist DS columns were prepared using a normal phase suspension method as shown in Figure 32 [43].

Fast desalting with excellent reproducibility could be carried out within 5 min using conventional HPLC system and TSKgel BioAssist DS Columns (4.6 mm ID and 10 mm ID) (Figure 33). All the proteins (see table below) eluted with the same retention time closer to void volume irrespective of their size (see the figures below), while salt and other small impurities eluted at longer retention time as a function of their size. Refractive index was used as a detector in this

SEC columns designed for desalting using a HPLC instrument can be useful for the desalting

Figure 32. Principle of the generation of pressure-stable polyacrylamide beads which can be packed in TSKgel BioAssist

of proteins and polynucleotides at analytical and semi-preparative scale.

study since salts do not have any chromophore.

DS columns.

166 Antibody Engineering

Figure 33. Desalting of proteins using a TSKgel BioAssist DS column with 4.6 mm ID or 10 mm ID packed with pressurestable polyacrylamide beads. The retention times of standard proteins and salt are shown.
