Numerous antibody drug conjugates (ADCs) are in clinical trials with most of them being used in cancer treatment with a toxin
payload (26). The major downstream challenge is separation of multiple species containing different levels of the attached
drug to the mAb. This results from the limited specificity for chemical conjugation of the drug to either exposed lysines
or cysteines (generated by limited reduction) on the mAb. To enable straightforward downstream processing of ADCs, two main
strategies have been used to generate more homogeneous molecules. These are the incorporation of additional free cysteines
that can then be chemically targeted specifically or to mutate out some of the native cysteines to serines so that they are
less available for coupling after a limited reduction reaction (27, 28).
mAB FRAGMENTS AND SINGLE DOMAIN mAbs
One approach to generate smaller and simpler BioTx for downstream processing is to replace full-length mAbs with antibody
fragments such as single-chain variable fragments (scFvs) and antigen-binding fragments (Fabs). Cimzia and Lucentis are two
recently approved Fabs produced in E. coli (29) and although no scFv has yet received regulatory approved, there are a large number of both types of Ab fragments in clinical
trials (30–32). Purification of these Ab fragments utilizes normal chromatographic procedures unless the H-chain is from the
VH3 IgG gene family in which case it will bind to protein A. After purification these small fragments are usually modified
to improve their pharmacokinetics using technologies, such as PEGylation. PEGylation dramatically changes the chromatographic
properties of proteins such that loading capacity is severely compromised thus requiring larger columns (33, 34). Additionally
PEGylation can dramatically increase the viscosity of concentrated drug substances.
In addition to mAb fragments, another approach to obtain smaller less complex antibody molecules is to use either Domain Abs
(human derived) or nanobodies (Llama derived), which are composed of a single variable domain of ~ 14kDa. These small single
domain antibodies can be easily captured from fermentation broth using Protein A since they can be derived from the VH3 IgG
gene family (35, 36). Subsequent downstream purification has been accomplished using standard chromatographic techniques (36).
Using viral vectors as BioTx for gene therapy offers many exciting opportunities to inactivate new targets. Their production
presents numerous challenges because of the complex nature of their composition and large size (2–5 x 106 kDa). They are usually composed of multiple proteins that encapsulate the nucleic acid payload. Additionally, there may be
empty capsids that do not contain the gene of interest but are similar in physicochemical properties and can be difficult
to separate from the desired product. CsCl density gradient centrifugation has long been the method of choice for their isolation;
however, for clinical testing, larger scale production has recently been accomplished using standard column chromatographic
methods. However, because of their large size, their binding capacity is usually very low and chromatographic media with normal
pore sizes used for proteins offer no advantages since the pores are not available. Smaller beads with more surface area is
the most effective manner to increase capacity (37,38). Other processes that are being utilized include tangential flow filtration
that takes advantage of the large particle size for separation from smaller cellular contaminants.
Vaccines can be produced using numerous technologies and have been generated against proteins, polysaccharide, and small molecule
targets. Pneumonia is the largest single infectious disease causing infant mortality (39). To date, three vaccines have been
licensed for treating diseases caused by pneumococcus-containing polysaccharides conjugated to proteins (40).
Because prophylactic vaccines are given to a large healthy population ranging from infants to the elderly, the regulatory
requirements during development and for licensure tend to be more stringent as compared with other biotherapeutic products.
This also results in more early investment and upfront development work for a vaccine candidate than for a model biotherapeutic
agent. Most commercial prophylactic vaccines are not well-characterized biologicals from a regulatory perspective because
of their inherent complexity and/or poorly understood mechanism of action. In addition, if an in vivo animal potency model is available, the results don't usually predict the human response to the product with regards to the
protein structure-immunogenecity/antigencity relationship. These reasons force sponsors to initiate extensive process characterization
studies and to lock the production processes early in the clinical development program (41).
One new approach to generate vaccines is to use virus–like particles (VLP) as potent immunostimulaters with covalent attachment
of many copies of the desired antigen to their surface (42). Similar to viral vectors, VLPs present unique challenges because
of their large size (2.5 x106 kDa). Q (14 kDa) is the most commonly used VLP. It is derived from the structural coat protein of this virus and it naturally
assembles into 180 copies of itself during expression in the cytoplasm of E. coli. Subsequent purification is accomplished by classical column chromatography techniques with size-exclusion chromatography
cited most frequently, which is not a desired step for scale-up (41). The challenges to develop alternative process steps
are similar to the viral vector BioTx because of low capacity for most resins as well as the fact that there may be heterogeneous
levels of antigen attached to the VLPs. Another new approach is the delivery of plasmid DNA to cells, which leads to the transcription
of antigens and a subsequent immune response (43).
An additional complexity is the fact that many vaccines are multivalent. The manufacturing processes for these vaccines involve
many different fermentation, purification, and conjugation trains for the polysaccharides and carrier proteins. Developing
processes that are similar between antigens can improve manufacturability and facility fit (44).