Biologics: the new class of drugs

1 in 4 new medicines is a biologic, as were 8 out of the 10 top-selling drugs globally in 2019. A biologic is a drug made from a biological source, rather than by chemical synthesis. These new drugs have superior selectivity and potency, but high structural complexity. 

What is a biologic? 

Biologics can be proteins, sugars, nucleic acids, or entire cells or tissues. In contrast to small molecule (SM) drugs such as paracetamol, aspirin or codeine, which are often taken in the form of pills or capsules, biologics are taken by injection. They must also be kept refrigerated. Biologics encompass a wide range of entities including monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs), peptides and recombinant proteins, glycoproteins, vaccines and blood components. 

Unlike SM drugs, which can be stored and taken as pills or capsules, biologics are administered via injection and require refrigeration to prevent degradation.

They have many advantages over SM drugs: they’re less hindered by traditional drawbacks such as antigenicity, as well as having higher affinity and specificity, minimising side effects and enhancing efficacy. They’re highly targeted by nature, leading to their use as treatments for cancers, infectious diseases and immune disorders, and are emerging as enabling tools in areas such as viral delivery and genome editing. The targeted distribution of biologics is particularly powerful against cancers, where these drugs selectively bind to cancer cell receptors and leave healthy cells unharmed. By comparison, SM drugs generally act on sites throughout the body, causing greater side effects.

What are the challenges involved in developing biologics?

However, biologics manufacture is far more complicated than the relatively rapid and low-cost manufacture of SM medicines. Most are made by genetically-engineered microorganisms in lengthy processes contained within bioreactor vessels, with reaction monitoring being key to ensuring correct drug action. This is because the structure of biologics are very large and highly complex, with inherent variability. This structural complexity makes their characterisation very challenging.

Biologics are highly complex: aspirin, an SM drug, measures only 180 daltons, whilst Pembrolizumab, an antibody for cancer treatment, measures 149,000 daltons.

Why is characterisation of biologics important?

For protein biologics, their structure determines their pharmacological activity and dictates their tendency towards aggregation (which decreases stability and shortens shelf-life). Accurate and thorough characterisation of a protein’s higher order structure (HOS), or the secondary, tertiary and quaternary structure of a protein, is critical, as it directly impacts on biologic efficacy, safety and stability. HOS affects many protein biophysical properties such as protein kinetics and particulate formation, and is responsible for correct protein folding and shape, which dictates drug functionality. An improper HOS can cause misfolding and an undesired protein shape, inhibiting receptor binding or exposure of immunogenic epitopes, and can lead to agglomeration.

How does IR spectroscopy probe biologics?

Whilst analysis of biologics in the UV-VIS and NIR regions involves selectivity and sensitivity challenges, mid-IR spectroscopy enables sensitive and label-free measurement and can be used to carry out simultaneous quantitation of multiple analytes within a bioreactor process. Futhermore, the mid-IR allows both qualitative and quantitative measurement of protein secondary structure, including α-helices, β-sheets, β-turns and structural disorder.

The molecular fingerprint mid-IR region has many advantages over UV-VIS and NIR analysis of biologics.

The vibrations of the polypeptide repeat units found within proteins result in nine characteristic group frequencies referred to as amide bands. Of particular importance are the Amide I (~1620-1700 cm-1), and Amide II (~1520-1580 cm-1) bands, which mainly stem from stretching vibrations of the amide C=O bond, and bending vibrations of the amide N-H bond, respectively. As both bonds are involved in the hydrogen bonding occurring between different elements in the secondary structure, the locations of the Amide I and Amide II bands are sensitive to the secondary structure content of a protein. They also give other information, such as the nature of protein unfolding.

Of the two bands, Amide I is the most intense absorption band. As well as indicating much information about secondary structure such as conformational changes and random coil formation, the Amide I band is often used for protein quantitation. However, when working with proteins in an aqueous environment the strong absorbance of the H-O-H bending band of water at ~1640 cm−1 overlaps with the Amide I band, requiring short path lengths of ~8 μm for conventional FTIR analysers and limiting LDR. An alternative is using ATR-IR probes, but these have long measurement times that hinder their effectiveness in a continuous bioreactor set-up, and, because they probe using an evanescent wave, only penetrate a short distance into a flow.

The mid-IR absorption spectrum of water. Its H-O-H bending band at ~1640 cm-1 makes analysis of the key protein Amide I band (~1620-1700 cm-1) difficult.

What can Quantum Cascade Laser (QCL) technology do in assisting in protein analysis?

Quantum cascade lasers (QCLs) were developed as a mid-IR light source over two decades ago. This type of laser involves inter sub-band transitions of electrons within the semiconductor conduction band, allowing them to generate spectral power densities several orders of magnitude greater than the thermal light sources conventionally used in FTIR spectrometers, such as globars. QCLs’ significantly high emission allows much longer optical paths for transmission measurements, facilitating the analysis of protein secondary structure in aqueous solution.

QCLs have a brightness magnitudes greater than that of globar and even synchrotron sources, allowing transmissive measurements and longer path lengths with higher sensitivity than FT-IR and ATR-IR.     

QCL-IR is a powerful analytical tool in many processes within biologic development: protein titre (quantitation), characterisation and purity measurement, molecular conjugation reaction monitoring, and bioprocess quality control testing of growth media and buffer.

ChemDetect™ and Culpeo® mid-IR analysers for real-time biophysical characterisation

The ChemDetect™ and Culpeo® Daylight DRS Solutions Mid-IR analysers.

Based on QCL technology, the ChemDetect™ and Culpeo® are in-line mid-IR analysers designed for real-time spectral analysis in aqueous workflows. They can be coupled in-line to a HPLC instrument, used with an injection system/autosampler or with TFF, or used online with a bioreactor set-up as a tool for process analytical technology (PAT) and bioprocess analytics. By using QCLs’ high emission, they’re able to carry out real-time, transmissive measurements of proteins in water solvent. Scan time is ~1/10 that of ATR-IR, with a 0.5-200 mg ml-1 linear dynamic range.

Quantitation of Lysozyme in water by measurement of its Amide I and II bands. Recorded on a Daylight Culpeo®, 1 s acquisition, 0.5 cm-1 step, without baseline correction or smoothing. R2 = 0.9996.

The ChemDetect™ is a pilot instrument designed for R&D phase research and QC processes. It’s highly flexible, with a controllable optical path length, and a flow cell usuable with a range of solvents to enable characterisations from protein titre to conjugation study. The ChemDetect™ is benchtop and stackable, with a built-in reference detector and dedicated software. Its embedded computer is programmed with chemical identification algorithms for unmatched speed and specificity. The ChemDetect™ is able to explore structural heterogeneities, probe molecular conformational dynamics, and perform simultaneous, real-time quantification and identification of multiple chemical species.

Its closely-related cousin, the Culpeo®, is a turn-key, GxP compliant platform tailored for specific industrial biologics applications, including mAb, ADC, glycoprotein, peptide drug and vaccine development. It enables easy method transfer, being designed with both precision and high throughput capacity to operate along the whole pharmaceutical process chain from discovery to analytical development, to QC and manufacturing. It supports regulatory affairs, meeting GMP and local FDA compliance for data capture, data transfer and data archival. Culpeo® has smart flow-cells for dedicated applications, as well as an OPC-UA interface for use with an internal software client to control of all aspects of data handling.

To find out more on the ChemDetect™ and see examples of its use in chemical and bioreactor monitoring, click here.

To find out more on the Culpeo® such as its use in biopharmaceutical and vaccine development, click here.

A video overview of the Culpeo®’s capabilities may also be viewed here.

Other Blogs/Articles that may be of interest

1.      Why is Mid-IR Light so Important?

2.      What is a Laser Spectrum Analyser and how does it differ from a Laser Wavemeter?

3.      Overview of emerging Quantum Cascade Laser applications

4.      Rapid Identification and quantification of microplastics using new QCL-IR imaging