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015-1790-3

12 Biopharmaceuticals

**Chapter 2**

**Provisional chapter**

**Characterization of Biopharmaceuticals Focusing on**

**Characterization of Biopharmaceuticals Focusing on** 

Biopharmaceuticals are highly complex molecules and also require high quality for safety and efficacy in human uses. For well-characterized products, the desired level of quality should be monitored and controlled during the manufacturing processes. A series of workflow for analytical characterization should be applied for product quality throughout those processes. In this chapter, several analytical techniques are introduced for assessing characteristics of biopharmaceuticals focusing on monoclonal antibodies (mAbs). Analytical characterization for primary structure was performed by mass spectrometry (MS), and assessment of post-translational modifications (PTMs) was done by conventional approaches. The analytical assessments were also done by multi-attribute method (MAM) approach using mass spectrometer (MS), and the performance of MAM

**Keywords:** biopharmaceutical, analytical characterization, primary structure, mass spectrometry, post-translational modification (PTM), multi-attribute method (MAM)

Biopharmaceuticals can be defined as protein drugs that are produced by recombinant DNA technology, such as hormones, enzymes, monoclonal antibodies, and fusion proteins used for therapeutic or diagnostic purposes [1]. The first biopharmaceutical, insulin, was introduced in 1982 [2], and since then, over 250 biopharmaceutical products are authorized for marketing in the two major regions, United States of America (USA) and Europe (EU). Those products can be classified into monoclonal antibodies (mAb), hormones, growth factors, vaccines, cytokines, blood factors, and others [3]. This trend with increasing number of biopharmaceuticals

> © 2016 The Author(s). Licensee InTech. 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.

© 2018 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.

DOI: 10.5772/intechopen.79107

**Antibody Therapeutics**

**Antibody Therapeutics**

http://dx.doi.org/10.5772/intechopen.79107

**Abstract**

**1. Introduction**

Woojeong Kim, Kui Hyun Kang and Jung-Keun Suh

Woojeong Kim, Kui Hyun Kang and Jung-Keun Suh

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

was compared to conventional approaches.

#### **Characterization of Biopharmaceuticals Focusing on Antibody Therapeutics Characterization of Biopharmaceuticals Focusing on Antibody Therapeutics**

DOI: 10.5772/intechopen.79107

Woojeong Kim, Kui Hyun Kang and Jung-Keun Suh Woojeong Kim, Kui Hyun Kang and Jung-Keun Suh

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79107

#### **Abstract**

Biopharmaceuticals are highly complex molecules and also require high quality for safety and efficacy in human uses. For well-characterized products, the desired level of quality should be monitored and controlled during the manufacturing processes. A series of workflow for analytical characterization should be applied for product quality throughout those processes. In this chapter, several analytical techniques are introduced for assessing characteristics of biopharmaceuticals focusing on monoclonal antibodies (mAbs). Analytical characterization for primary structure was performed by mass spectrometry (MS), and assessment of post-translational modifications (PTMs) was done by conventional approaches. The analytical assessments were also done by multi-attribute method (MAM) approach using mass spectrometer (MS), and the performance of MAM was compared to conventional approaches.

**Keywords:** biopharmaceutical, analytical characterization, primary structure, mass spectrometry, post-translational modification (PTM), multi-attribute method (MAM)

## **1. Introduction**

Biopharmaceuticals can be defined as protein drugs that are produced by recombinant DNA technology, such as hormones, enzymes, monoclonal antibodies, and fusion proteins used for therapeutic or diagnostic purposes [1]. The first biopharmaceutical, insulin, was introduced in 1982 [2], and since then, over 250 biopharmaceutical products are authorized for marketing in the two major regions, United States of America (USA) and Europe (EU). Those products can be classified into monoclonal antibodies (mAb), hormones, growth factors, vaccines, cytokines, blood factors, and others [3]. This trend with increasing number of biopharmaceuticals

© 2016 The Author(s). Licensee InTech. 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. © 2018 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.

on the market results in gaining interest for drug development industry, and biopharmaceuticals are considered as fast growing and promising area for drug development [3–6].

**2. Structural characterization for identity**

deletion (ΔK), or Gln/Glu cyclization (pE) (**Figure 1**).

**2.2. Subunit mass determination of mAb**

pE: pyro-glutamate.

The molecular weight of a protein is an important parameter in the physicochemical properties of the protein. MS with high resolution and accuracy, such as matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) or electrospray ionization quadrupole time-of-flight (ESI-QTOF), has become a reliable and sensitive technique for proteins and peptides to determine the molecular weight [26, 27]. Other high resolution mass spectrometers, Fourier transform ion cyclotron resonance (FTICR) and orbitrap MS, have been recently used for the intact mass analysis [28–30]. The workflow of ESI was further extended to native MS, having the capability to investigate intact mAb for structural analysis and heterogeneities from PTM [31, 32]. Applying intact mass analysis, it not only gives molecular mass of the intact protein for comparison with the theoretical mass but also provides quick information about primary structure of protein or sequence variants, such as isoforms, truncation, muta-

Characterization of Biopharmaceuticals Focusing on Antibody Therapeutics

http://dx.doi.org/10.5772/intechopen.79107

17

ESI-QTOF is widely used for measuring intact mass because of high resolution, mass accuracy, and easy connection to high performance liquid chromatography (HPLC) for online analysis [34]. ESI produces multiply charged ions, and those ions can be deconvoluted for molecular mass of proteins [34]. Typical results of mass determination for mAbs, bevacizumab, rituximab, and trastuzumab using ESI-QTOF are shown in **Figure 1**. MS spectra in inset of **Figure 1** show multiply charged ions characterized by ESI-spectrum, a number of peaks corresponding a statistical distribution of different charge states. The accuracy was less than 50 ppm, providing tools for the identification of PTMs, glycosylation, C-terminal Lysine

Subunits or fragments of mAbs can be obtained by reduction of disulfide bonds or proteolysis to reduce complexities for large size mAbs [36, 37]. Chemical reduction of mAb's disulfide bond generates free heavy chains and light chains, having ~50 kDa and ~25 kDa molecular mass, respectively. Recently, a new protease IdeS (Immunoglobulin G-degrading enzyme of *Streptococcus pyogenes*), specifically cleaving between the two consecutive glycine residues of the hinge region, has been described for mAb fragmentations [29, 37–39]. IdeS treatment of

**Figure 1.** Intact mass of mAbs determined by deconvolution using ESI-QTOF. Inserts show charge envelope having multiply charged ions. Glycan structures (G0F, G1F, and G2F) were adapted from [35]. ΔK: C-terminal Lys deletion and

tions, addition of signal sequences, or PTMs including glycosylations [31–33].

**2.1. Intact mass determination of mAb**

The approval of mAb-related products is dramatically increased in the recent years [6, 7]. Over 90 mAb-related products are approved by European Medicines Agency (EMA) and US Food and Drug Administration (FDA). Those can be classified into mAb, Fc-fusion, Fab, antibody-drug conjugate (ADC), bispecific mAb (bsAb), and bispecific T cell engager (BiTE). Among them, mAbs are the major product, consisting of 77% of total. Others represent rest 23% of total, Fc-fusion (12%), ADC (5%), Fab (3%), bsAb (2%), and BiTE (1%), respectively. After the first approval of full-length mAb in 1998, mAbs are major product in the biopharmaceutical industry. This increasing number gives high revenue for pharmaceutical companies, and seven mAb-related products are positioned in top 10 drugs in the world, 2017, including Humira, Enbrel, Rituxan, Remicade, AVASTIN, Herceptin, and Lantus [8].

Mylotarg is the first approved ADC in 2000, which combined a mAb targeting leukemic blast cells with a bacterial toxin (calicheamicin) [7, 9]. ADC is a complex generated between a mAb and small molecule or a peptide, and mAb gives the selective delivery for targeting of cytotoxic drugs [1, 9–11]. Since the first approval, four additional ADC products are approved in Europe and USA. bsAb has two different antigen binding sites recognizing two different epitopes in a single mAb, and this dual specificity gives more specific targeting and higher efficacy [12–14]. Currently, three bsAbs are approved by EMA or US FDA. The first bsAb, Removab, was approved in 2009 but voluntarily withdrawn in 2013. Fc-fusion proteins are fusions of the IgG Fc domain with a desired linked protein, enhancing pharmacokinetic properties (serum half-life) and pharmacodynamics properties (ADCC and CDC) [6, 15]. Following the first approval of Fc-fusion protein, Enbrel in 1998, eight Fc-fusion proteins are authorized for the marketing in the region of Europe and USA.

Biosimilars, known as follow-on biologics, which follow termination of patent protection of original biopharmaceutical products, are developed and approved since 2006. Following the first approval from EMA, over 35 biosimilars are authorized for the European market and over 20 biosimilars are approved from FDA since 2015 [16–18]. A biosimilar is a biological product that is highly similar to and has no clinically meaningful differences from an existing FDA-approved reference product in terms of safety, purity, and potency (safety and effectiveness) [19]. Structural and clinical similarities are to be proven for the biosimilar authorization.

Biopharmaceuticals are highly complex molecules compared to small molecule drugs and should be monitored and controlled during the manufacturing processes for well-characterized products [20–22]. The characterization of biopharmaceuticals is challenging, which utilize the state-of-the-art technology to meet the international harmonized guidelines, Q5E and Q6B [23, 24]. For proper characterization, critical quality attributes (CQAs) have to be defined and evaluated that may impact on safety, purity, and potency. CQA is defined by ICHQ8(R2) as a physical, chemical, biological, or microbiological property of characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality [25].

Within this chapter, methodologies for in-depth physicochemical characterization are introduced for recombinant biopharmaceuticals mainly focused on mAbs. Analytical characterization for primary structure was performed by mass spectrometry, and assessment of post-translational modifications (PTMs) was done with conventional and multi-attribute method (MAM) approaches. The performance of MAM was compared to conventional approach.
