Monoclonal antibodies (mAbs) dominate the biotherapeutic market [1] with IgG1s2, [[2], [3]], followed by IgG4s3 [[2], [3]], as treatments for various life-threatening diseases, particularly cancers [3]. They have been shown to offer improved specificity and longer half-lives compared to the traditional pharmaceutical drugs. However, mAbs are known to have multiple critical quality attributes (CQAs) and are known to be prone to fragmentation and clipping [4], [5], [6]. Fragmentation of a mAb involves non-enzymatic [7] or enzymatic [8] cleavage of the peptide bond and this can occur during cell culture [9], [10], [11], [12], purification [9], formulation [9,10], storage [10,11] or administration [9]. Non-enzymatic fragmentation may also occur at acidic or basic pH [9], high temperature [10], under oxidative stress [8], or when exposed to excessive metal ions [8] or light [13]. Thus, formation of the various fragments (100–120 kDa) or clips (20–50 kDa) [10] depends on the mechanism and site of fragmentation.

Furthermore, proteases created during protein expression are known to cause enzymatic fragmentation [14]. Fragmentation is considered a CQA as it may lead to change in the higher mAb order structure and thereby impact efficacy [5], [6], [7], [8] and hence is considered one of the degradation pathways for mAb products [14]. However, fragmentation is often neglected, as it is difficult to assess [15] and control [6,14]. Downstream purification of fragments is non-trivial due to the similar biophysical characteristics of the fragment specie and the parent mAb [16]. Also, fragments encompass a range of species [17]. Similar charge profile and/or binding mechanism of some of these fragment species makes it difficult to separate them using traditional chromatography set ups, solely based on charge or mass separation.

Conventional purification process design does not target fragment removal. Researchers have used various chromatographic resins such as IEX [18,19,20], HIC [18], MMC [21], and affinity [19,20] for separating fragments (Table 1). However, most studies suffer from poor resolution [[19], [20], [22]], narrow applicability (targeting a single type of mAb) [[20], [22], [23]] or demonstrate only separation of low molecular weight fragment [[19], [20], [22], [23], [24]] (Table 2). Preparative size exclusion chromatography (SEC) can also be used for removal of fragments, but SEC suffers from dilution of the sample and requirement of large column sizes at preparative scales. In recent times, multimodal chromatography (MMC) resins have gained popularity due to the higher resolution and unique selectivity that they offer for mAb separation [21]. Their multiple modes of interactions include electrostatic [22,25], hydrophobic [22,26,27], affinity [28], and hydrogen bonding [25,27] modes, as have been catalogued in recently published top-down analysis [29], [30], [31]. The binding interface is predominantly modulated by the hinge region of the mAbs, followed by the domains with higher surface hydrophobicity (like loops such as complementarity-determining regions) [[29], [31], [32]]. Thus, novel selectivity is subject to domain hydrophobicity and resin ligand chemistry. Further, mAbs display unique binding under optimal conditions due to differences in the hydrophobic content compared to similarly charged fragments [29], [30], [31].

The objective of the current study was to develop a robust purification method for removal of fragments and clipped species. The study utilized a novel multimodal chromatography method to achieve complete removal of a range of mAb fragments (25–120 kDa). The developed method exploited the differential binding of fragments to the resin as compared to intact mAb, under optimal ionic strengths. Binding of the fragments was hindered due to incomplete or lack of hinge region, hence, aiding the separation of the fragments from the complete mAb. It was then integrated in two different purification platforms to demonstrate the utility for the two IgG1s (mAbs A and B) and 2 IgG4s (mAbs R1 and R2). Adequate removal of the various host cell impurities such as host cell proteins (<10 ppm) and host cell DNA (<5 ppb) has also been demonstrated. Further, the platform was able to deliver adequate removal of HMWI (<1 %) and a 30 % clearance of the acidic charge variant. A step yield of >95 % and purity of >99.0 % has been achieved. The proposed single step has been shown to deliver what the polishing chromatography and intermediate purification chromatography steps deliver in a traditional mAb platform.