Quality Control and Analytical Methods for Recombinant Protein Biopharmaceuticals

Recombinant Protein Biopharmaceuticals

With the maturation and application of genetic engineering technology, it is now possible to design, modify, and express proteins as needed, leading to a diverse array of recombinant proteins. Recombinant protein biopharmaceuticals, characterized by their strong specificity, low dosage requirements, and minimal side effects, play a crucial role in the prevention and treatment of various diseases. Consequently, quality control for these products becomes especially vital.

For these proteins, the initial data typically obtained is their amino acid sequence. Amino acid analysis can leverage this limited information to analyze recombinant proteins, determining their amino acid content and composition, as well as protein concentration and absorption coefficients.

Amino Acid Content and Composition Analysis

Recombinant protein products, expressed and purified through genetic engineering techniques, face the critical challenge of ensuring that their structure and sequence align with the intended design. Amino acid composition analysis plays a pivotal role in quality control by determining the content and proportion of various amino acid residues that make up the protein. This analysis is an essential step before determining the full sequence of the protein. In situations where rapid determination of the entire protein sequence is not feasible, amino acid composition analysis serves as a robust confirmation of the primary structure of the protein.

For accurate analysis, the purity of the protein is typically required to be greater than 95%. The hydrolysis method, commonly using acid hydrolysis, is employed for amino acid analysis, with acid removal and volume adjustment following the standard amino acid analysis methods. When calculating the relative molar ratios of various amino acids, selecting a reference amino acid with a recovery rate of 100% is crucial. The choice of the reference amino acid depends on the amino acid composition of the specific protein. Generally, a stable amino acid in hydrolysis, easily separable by ion exchange chromatography, or one with a higher content in the entire protein is chosen.

Discrepancies between experimentally measured relative molar ratios and theoretical values arise due to various factors. Hydrolysis can cause significant destruction of unstable amino acids (such as serine and threonine), leading to lower measured values compared to theoretical values. Additionally, amino acids with large spatial hindrance, like isoleucine, may be challenging to hydrolyze, resulting in lower measured values. Furthermore, different amino acids may be affected to varying degrees under experimental conditions, leading to slight deviations from theoretical values even when calculated based on the reference amino acid.

Currently, amino acid composition analysis is not a routine indicator for quality control of recombinant protein drugs. However, the information on the composition and proportions of various amino acids obtained through analysis holds significance in distinguishing differences between biotechnologically expressed proteins and their natural counterparts. It aids in identifying and confirming whether mutations have occurred as expected, providing valuable guidance for quality control and structural confirmation. To obtain more precise structural information, a combination of N-terminal amino acid sequencing, peptide mapping analysis, circular dichroism analysis, DNA sequencing, or mass spectrometry analysis is recommended.

Accurate Quantification of Protein Content

Protein content determination is a crucial parameter in the quality control of recombinant protein drugs. Accurate results from protein content assays hold significant implications for product packaging, specific activity calculations, limiting the control of residual impurities, and other physicochemical property assessments. Therefore, achieving precise protein quantification for recombinant protein drugs remains a key challenge in the quality control of biotechnological pharmaceuticals.

Currently, widely adopted methods such as Lowry, BCA, and Bradford assays rely on the reaction of specific amino acids or functional groups in proteins, coupled with the use of standard or control samples for quantification. While these methods generally meet the requirements for recombinant protein quantification, accurate calibration of standard or control samples is crucial to ensure the reliability and accuracy of protein content determination. Kjeldahl nitrogen determination or amino acid analysis is commonly used for standard or control sample calibration in protein content determination. Both methods are recognized as "gold standards," although Kjeldahl nitrogen determination may have limitations due to the requirement for a larger sample volume, restricting its application to some extent. In cases where samples are difficult to obtain or have limited volume, the advantage of using amino acid analysis for quantification becomes apparent.

The continuous development of biotechnology has led to the emergence of complex proteins with structural changes caused by glycosylation and PEGylation, rendering traditional content determination methods ineffective. Amino acid analysis is considered a viable method for the quantitative analysis of these proteins.

Amino acid analysis involves the absolute quantification of various amino acids constituting a protein, offering an objective, minimally interfered, and highly automated approach with broad applicability. In protein content determination, the assumed knowledge of the amino acid sequence, polysaccharide structure, and relative molecular weight of the relevant protein is essential. Subsequently, the sample undergoes amino acid analysis to obtain the content of each amino acid, followed by calculating the mass of the analyzed protein sample using the formula: Protein Content (W) = Average [Amino Acid Content / Number of this amino acid in the protein] × Relative molecular weight of the protein.

Given the variations in the hydrolysis of different amino acids, it is recommended to use amino acids with good recovery, such as Ala, Ser, Leu, Ile, Thr, Tyr, when calculating. For glycosylated proteins with high sugar content, alternative methods like mass spectrometry, HPLC, or ELISA should be considered for quantification, as the limitations of amino acid analysis may come into play.

Calculation of Absorption Coefficient in Protein Products

The absorption coefficient is a unique constant for protein products and serves as the foundation for establishing relevant standards, as recommended by ICH Q6B. The absorption coefficient E₀.₁% represents the absorbance of a 1 mg/ml or 0.1% mass/volume protein solution in a 1 cm cuvette. This coefficient can be calculated based on the amino acid analysis results of the corresponding protein or obtained from the protein's sequence information. While the latter is more convenient, it has practical limitations. On one hand, most recombinant protein drugs are fusion proteins or mutants obtained through genetic engineering, making it challenging to determine their absorption coefficients. On the other hand, the addition of excipients and additives in protein formulations can alter the sample's pH, affecting the ionization state of tyrosine residues and subsequently influencing protein absorbance. Therefore, accurate absorption coefficients are often determined using amino acid analysis.

In the calculation, the protein content is determined using amino acid analysis, and then the absorption coefficient is calculated using the absorbance at UV280 (A280) determined in the formulation with a known optical path length (L). The formula used for the calculation is as follows:

Absorption Coefficient= A280/ (L × W / V)

Here, W represents mass, and V represents volume.

Factors influencing absorbance include the amino acid composition of the protein and the microenvironment in which the protein is situated. Phenylalanine, tyrosine, tryptophan, and cysteine compositions are the primary influencing factors. In practical determinations, a detailed analysis based on the specific characteristics of the protein is recommended. Additionally, combining other methods can provide a comprehensive approach to determining the absorption coefficient.